Polymicrobial formulations for enhancing plant productivity

ABSTRACT

The present invention relates to eco-friendly compositions and methods for providing plant growth enhancing formulations comprising mixtures of microbial isolates. In particular, numerous bacterial and fungal strains were isolated from a variety of soil types, from rhizospheres and from root nodules of leguminous plants, in designed combinations, for providing plant growth and plant productivity enhancing formulations. These specifically designed polymicrobial formulations would further provide protection against plant pathogens lowering the need for nitrogen containing fertilizers, solubilize minerals, protect plants against pathogens, and make available to the plant valuable nutrients, such as phosphate, thus reducing and eliminating the need for using chemical fertilizers and chemical pesticides.

FIELD OF THE INVENTION

The present invention relates to eco-friendly compositions and methodsfor providing plant growth enhancing formulations comprising mixtures ofbeneficial microbial isolates. In particular, numerous bacterial andfungal strains were isolated from a variety of soil types, fromrhizospheres and from root nodules of leguminous plants, in designedcombinations, for providing plant growth and plant productivityenhancing formulations. These specifically designed polymicrobialformulations would further provide protection against plant pathogenslowering the need for nitrogen containing fertilizers, solubilizeminerals, protect plants against pathogens, and make available to theplant valuable nutrients, such as phosphate, thus reducing andeliminating the need for using chemical pesticides and chemicalfertilizers.

BACKGROUND

For a long time plant biologists knew that a number of species ofrhizobacteria (bacteria naturally occurring in the plant rhizosphere)beneficially affected plant growth albeit by employing differentmechanisms. These mechanisms include: 1) contribution to the nitrogeneconomy of the plant by fixing atmospheric nitrogen (N₂); 2) producinggrowth stimulant compounds such as various auxins; and 3) inhibiting anumber of plant pathogenic bacteria and fungi. Therefore, it wasbelieved that naturally occurring rhizobacteria contributed not only toincreased plant growth but was also to protect plants against pathogens.

Numerous types of rhizobacteria have been isolated and appliedexogenously to plants as growth enhancers with a stated goal ofminimizing the need for environmentally harmful chemical pesticides andfertilizers. However, due to the complexity of the rhizosphere,bacterial additions to the soil do not result in the types of increasesin plant growth or productivity expected by the plant growers.

Thus it would greatly benefit our environment to have naturally derivedmicrobial products for increasing plant growth and for reducing the needfor applications of pesticides.

SUMMARY OF THE INVENTION

The present invention relates to eco-friendly compositions and methodsfor providing plant growth enhancing formulations comprising mixtures ofmicrobial isolates. In particular, numerous bacterial and fungal strainswere isolated from a variety of soil types, from rhizospheres and fromroot nodules of leguminous plants, in designed combinations, forproviding plant growth and plant productivity enhancing formulations.These specifically designed polymicrobial formulations would furtherprovide protection against plant pathogens lowering the need fornitrogen containing fertilizers, solubilize minerals, protect plantsagainst pathogens, and make available to the plant valuable nutrients,such as phosphate, thus reducing and eliminating the need for usingchemical pesticides and chemical fertilizers.

The present invention provides exemplary isolates of soil bacterialstrains and fungal strains as described herein.

Specifically, the present invention provides an isolated Ensifermeliloti FD bacterial strain having accession number _(——————).

The present invention provides an isolated Rhizobium trifolii FDbacterial strain having accession number _(——————).

The present invention provides an isolated Azorhizobium caulinodans KNbacterial strain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG1 bacterialstrain having accession number _(——————).

The present invention provides an isolated Azorhizobium sp. RLG2bacterial strain having accession number _(——————).

The present invention provides an isolated Azorhizobium sp. RLG3bacterial strain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG4 bacterialstrain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG5 bacterialstrain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG6 bacterialstrain having accession number _(——————).

The present invention provides an isolated Azorhizobium sp. RLG7bacterial strain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG8 bacterialstrain having accession number _(——————).

The present invention provides an isolated Azorhizobium sp. RLG9bacterial strain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG10 bacterialstrain having accession number _(——————).

The present invention provides an isolated Rhizobium sp. RLG11 bacterialstrain having accession number _(——————).

The present invention provides an isolated Trichoderma virens 3107fungal strain having accession number _(——————).

The present invention provides an isolated Trichoderma viride LK fungalstrain having accession number _(——————).

The present invention provides an isolated Trichoderma viride 3116fungal strain having accession number _(——————).

The present invention provides an isolated Trichoderma harzianum 3147fungal strain having accession number _(——————).

The present invention provides an isolated Trichoderma harzianum Gfungal strain having accession number _(——————).

The present invention provides an isolated Trichoderma harzianum LKfungal strain having accession number _(——————).

The present invention provides an isolated Trichoderma longibrachiatum3108 fungal strain having accession number _(——————).

The present invention provides an isolated Bacillus sp. LK bacterialstrain having accession number _(——————).

The present invention provides an isolated Bacillus subtilis LKbacterial strain having accession number _(——————).

The present invention provides an isolated Pseudomonas fluorescens CAbacterial strain having accession number _(——————).

The present invention provides an isolated Azospirillum CA bacterialstrain having accession number _(——————).

The present invention provides an isolated Acetobacter sp. LK bacterialstrain having accession number _(——————).

The present invention provides an isolated Rhizobium phaseoli CAbacterial strain having accession number _(——————).

The present invention provides an isolated Bradyrhizobium japonicum CAbacterial strain having accession number _(——————).

The present invention provides an isolated Rhizobium meliloti FDbacterial strain having accession number _(——————).

The present invention provides an isolated Paenibacillus brasiliensis172 bacterial strain having accession number _(——————).

The present invention provides a microbial formulation, wherein saidformulation comprises at least two microbial isolates. In oneembodiment, the two microbial isolates are isolated from soil. In oneembodiment, the two microbial isolates are isolated from a root nodule.In one embodiment, the two microbial isolates consist of a soilmicrobial isolate and a root nodule isolate. In one embodiment, saidformulation comprises at least seven microbial isolates. In oneembodiment, said formulation comprises at least twenty-one microbialisolates. In one embodiment, said formulation comprises at least sevenmicrobial isolates. In one embodiment, said formulation comprises atleast twenty-one microbial isolates. In one embodiment, said formulationcomprises at least twenty-one microbial isolates. In one embodiment,said formulation consists of twenty-one microbial isolates. In oneembodiment, said formulation comprises up to at least forty microbialsoil isolates. In one embodiment, said formulation comprises up to atleast forty microbial soil isolates. In one embodiment, said formulationcomprises at least seven and up to forty microbial soil isolates. In oneembodiment, said microbial isolate is selected from the group consistingof a bacterial isolate and a fungal isolate. In one embodiment, saidfungal isolate is selected from the group consisting of a Trichodermavirens 3107 fungal strain having accession number _(——————), aTrichoderma viride G fungal strain having accession number _(——————), aTrichoderma viride LK fungal strain having accession number _(——————), aTrichoderma harzianum 3147 fungal strain having accession number_(——————), a Trichoderma harzianum G fungal bacterial strain havingaccession number _(——————), a Trichoderma harzianum LK fungal strainhaving accession number _(——————), a Trichoderma longibrachiatum 3108fungal strain having accession number _(——————), In one embodiment, saidbacterial soil isolate is selected from the group consisting of aBacillus sp. RG-S bacterial strain having accession number _(——————), anEnsifer meliloti FD bacterial strain having accession number _(——————),a Rhizobium trifolii FD bacterial strain having accession number_(——————), an Azorhizobium caulinodans KN bacterial train havingaccession number _(——————), a Rhizobium sp. RLG1 bacterial strain havingaccession number _(——————), an Azorhizobium sp. RLG2 bacterial strainhaving accession number _(——————), an Azorhizobium sp. RLG3 bacterialstrain having accession number _(——————), a Rhizobium sp. RLG4 bacterialstrain having accession number _(——————), a Rhizobium sp. RLG5 strainhaving accession number _(——————), a Rhizobium sp. RLG6 bacterial strainhaving accession number _(——————) , Azorhizobium sp. RLG7 bacterialstrain having accession number _(——————), a Rhizobium sp. RLG8 bacterialstrain having accession number _(——————), an Azorhizobium sp. RLG9bacterial strain having accession number _(——————), a Rhizobium sp.RLG10 bacterial strain having accession number _(——————), a Rhizobiumsp. RLG11 bacterial strain having accession number _(——————), a Bacillussp. LK bacterial strain having accession number _(——————), a Pseudomonasfluorescens CA bacterial strain having accession number _(——————), anAzospirillum CA bacterial strain having accession number _(——————), anAcetobacter sp. LK bacterial strain having accession number _(——————), aRhizobium phaseoli CA bacterial strain having accession number_(——————), a Bradyrhizobium japonicum bacterial strain having accessionnumber _(——————), a Rhizobium meliloti FD bacterial strain havingaccession number _(——————), a Paenibacillus brasiliensis 172 bacterialstrain having accession number _(——————), a Paenibacillus peoriaebacterial strain having accession number BD-62, a Paenibacillus polymyxabacterial strain having accession number B37-A. In one embodiment, saidmicrobial soil isolate is selected from the group consisting of aBacillus sp. RG-S bacterial strain having accession number _(——————), anEnsifer meliloti FD bacterial strain having accession number _(——————),a Rhizobium trifolii FD bacterial strain having accession number_(——————), an Azorhizobium caulinodans KN bacterial train havingaccession number _(——————), a Rhizobium sp. RLG1 bacterial strain havingaccession number _(——————), an Azorhizobium sp. RLG2 bacterial strainhaving accession number _(——————), an Azorhizobium sp. RLG3 bacterialstrain having accession number _(——————), a Rhizobium sp. RLG4 bacterialstrain having accession number _(——————), a Rhizobium sp. RLG5 strainhaving accession number _(——————), a Rhizobium sp. RLG6 bacterial strainhaving accession number _(——————) , Azorhizobium sp. RLG7 bacterialstrain having accession number _(——————), a Rhizobium sp. RLG8 bacterialstrain having accession number _(——————), an Azorhizobium sp. RLG9bacterial strain having accession number _(——————), a Rhizobium sp.RLG10 bacterial strain having accession number _(——————), a Rhizobiumsp. RLG11 bacterial strain having accession number _(——————), aTrichoderma virens 3107 fungal strain having accession number _(——————),a Trichoderma viride LK fungal strain having accession number _(——————),a Trichoderma viride 3116 fungal strain having accession number_(——————), a Trichoderma harzianum 3147 fungal strain having accessionnumber _(——————), a Trichoderma harzianum G fungal strain havingaccession number _(——————), a Trichoderma harzianum LK fungal strainhaving accession number _(——————), a Trichoderma longibrachiatum 3108fungal strain having accession number _(——————), a Bacillus sp. LKbacterial strain having accession number _(——————), a Pseudomonasfluorescens CA bacterial strain having accession number _(——————), anAzospirillum CA bacterial strain having accession number _(——————), anAcetobacter sp. LK bacterial strain having accession number _(——————), aRhizobium phaseoli CA bacterial strain having accession number_(——————), a Bradyrhizobium japonicum bacterial strain having accessionnumber _(——————), a Rhizobium meliloti FD bacterial strain havingaccession number _(——————), a Paenibacillus brasiliensis 172 bacterialstrain having accession number _(——————), a Paenibacillus peoriaebacterial strain having accession number BD-62, a Paenibacillus polymyxabacterial strain having accession number B37-A. In one embodiment, saidmicrobial soil isolate is selected from the group consisting of aBacillus sp. LK bacterial strain having accession number _(——————), anBacillus subtilis LK bacterial strain having accession number _(——————),a Rhizobium trifolii FD bacterial strain having accession number_(——————), an Azorhizobium caulinodans KN bacterial train havingaccession number _(——————), a Pseudomonas fluorescens bacterial strainhaving accession number _(——————), an Azospirillum CA bacterial strainhaving accession number _(——————), an Acetobacter sp. LK bacterialstrain having accession number _(——————), a Rhizobium phaseoli CAbacterial strain having accession number _(——————), a Bradyrhizobiumjaponicum bacterial strain having accession number _(——————), anAzorhizobium caulinodans KN bacterial strain having accession number_(——————) , Trichoderma virens 3107 fungal strain having accessionnumber _(——————), a Trichoderma viride 3116 fungal strain havingaccession number _(——————), a Trichoderma harzianum 3147 fungal strainhaving accession number _(——————), a Trichoderma longibrachiatum 3108fungal strain having accession number _(——————). In one embodiment, saidmicrobial formulation further comprises a carrier, such that themicrobial formulation of the present inventions are delivered to a seedor plant in a manner to promote growth and productivity, such asgermination, yield, and the like. It is not meant to limit the type ofcarrier. Indeed, a variety of carriers are contemplated including butnot limited to a liquid, a solid and a combination of a liquid and asolid carrier. In a preferred embodiment, the carrier is a liquidcomprising water. In some embodiments, a carrier comprises a microbialgrowth medium. In some embodiments, a carrier further comprises humicacid, minerals, artificial compounds, particles, such as beads, powdersor granules, and the like. In some embodiments, a particle comprises aresin, clay, a biodegradable compound, and the like. In one embodiment,a bead comprises polymethyl methacrylate (PMMA).

In one embodiment, minerals comprise elements, such as Ca, Mg, and thelike. In some embodiments, minerals are compounds such as NH₄NO₃,KH₂PO₄, K₂HPO₄, MgSO₄, Ca(NO₃), KCl, KH₂PO₄, MgSO₄, CaSO₄, and the like.In one embodiment, minerals comprise trace elements, including but notlimited to any trace mineral comprising a trace element of benefit to amicrobe and a plant. Examples of such trace minerals are H₂BO₃L, ZnSO₄,CuSO₄, MnCl₂, Na₂MoO₄, et cetera. Both synthetic and natural compoundsare contemplated as components of formulations of the presentinventions, in particular for providing a benefit to a microbe or aplant, such as providing pathogen resistance, fungal resistance,reducing weeds, for example, an herbicide, a pesticide, a fungicide, aplant growth regulator, and for enhancing the effect of the microbialcompound, for example, an encapsulation agent, a wetting agent, adispersing agent, and the like. In one embodiment, a herbicide includesbut is not limited to imazethapyr, 2,2-dichloropropionic acid,glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), etc., andderivatives thereof. In one embodiment, a pesticide includes but is notlimited to O, S-dimethyl acetylphos-phoramidothioate (acephate),carbamate, carbaryl, chrlopyrifos-methyl, dicrotophos, indoxacarb,2-(dimethoxyphosphinothioylthio) (malathion), methomyl, methoxyfenozide,methyl parathion, pyrethrins, synthetic pyrethroids (such as bifenthrin,cypermethrin and the like), pyrethroids, protenophos, phorate, spinosyn,dimethylN,N′-[thiobis[(methylimino)carbonyloxy]]-bis[ethanimidothioate](thiodicarb),and derivatives thereof. In one embodiment, a plant growth regulatorincludes but is not limited to 2,2-dichloropropionic acid, and the like.

In one embodiment, said liquid carrier comprises water and humic acid.In one embodiment, said humic acid ranges from a concentration of0.0001%-60% volume/volume. In one embodiment, said humic acid is 12%volume/volume. In one embodiment, said liquid carrier comprises amineral solution. It is not meant to limit the mineral solution, indeeda variety of minerals are contemplated for use including but not limitedto individual minerals such as Ca, Co, Mg, Fe, etc., and mineralcompounds such as CoCL₂, H₃BO₃, MnCL₂, ZnSO₄, CuSO₄, H₂MoO₄, MgSO₄,K₂HPO₄, KH₂PO₄, CaCL₂, FeC₆H₅O₇, etc. In one embodiment, said liquidcarrier has a pH ranging from 5-9. In one embodiment, said liquidcarrier has a pH of 7.0. The concentration of microbes in a liquidcarrier may alter depending upon the carrier and the target amount ofplant enhancing characteristics. However, any concentration that willachieve plant-enhancing characteristics is desired. In some embodiments,said microbial isolate concentration in the liquid carrier ranges from10¹⁰-10¹⁷ microbes per milliliter of liquid. In one embodiment, saidmicrobial isolate concentration in the liquid carrier is selected fromthe group consisting of 10¹⁰, 10¹⁴, 10¹⁵, and 10¹⁷.

The present invention provides a method for enhancing plant growth,comprising, a) providing, i) a microbial formulation comprising amicrobial soil isolate, wherein said microbial soil isolate is selectedfrom the group consisting of a Bacillus sp. RG-S bacterial strain havingaccession number _(——————), an Ensifer meliloti FD bacterial strainhaving accession number _(——————), a Rhizobium trifolii FD bacterialstrain having accession number _(——————), an Azorhizobium caulinodans KNbacterial train having accession number _(——————), a Rhizobium sp. RLG1bacterial strain having accession number _(——————), an Azorhizobium sp.RLG2 bacterial strain having accession number _(——————), an Azorhizobiumsp. RLG3 bacterial strain having accession number _(——————), a Rhizobiumsp. RLG4 bacterial strain having accession number _(——————), a Rhizobiumsp. RLG5 strain having accession number _(——————), a Rhizobium sp. RLG6bacterial strain having accession number _(——————) , Azorhizobium sp.RLG7 bacterial strain having accession number _(——————), a Rhizobium sp.RLG8 bacterial strain having accession number _(——————), an Azorhizobiumsp. RLG9 bacterial strain having accession number _(——————), a Rhizobiumsp. RLG10 bacterial strain having accession number _(——————), aRhizobium sp. RLG11 bacterial strain having accession number _(——————),a Trichoderma virens 3107 fungal strain having accession number_(——————), a Trichoderma viride LK fungal strain having accession number_(——————), a Trichoderma viride 3116 fungal strain having accessionnumber _(——————), a Trichoderma harzianum 3147 fungal strain havingaccession number _(——————), a Trichoderma harzianum G fungal strainhaving accession number _(——————), a Trichoderma harzianum LK fungalstrain having accession number _(——————), a Trichoderma longibrachiatum3108 fungal strain having accession number _(——————), a Bacillus sp. LKbacterial strain having accession number _(——————), a Pseudomonasfluorescens CA bacterial strain having accession number _(——————), anAzospirillum CA bacterial strain having accession number _(——————), anAcetobacter sp. LK bacterial strain having accession number _(——————), aRhizobium phaseoli CA bacterial strain having accession number_(——————), a Bradyrhizobium japonicum bacterial strain having accessionnumber _(——————), a Rhizobium meliloti FD bacterial strain havingaccession number _(——————), a Paenibacillus brasiliensis 172 bacterialstrain having accession number _(——————), a Paenibacillus peoriaebacterial strain having accession number BD-62, a Paenibacillus polymyxabacterial strain having accession number B37-A; and ii) a plant, andapplying said microbial formulation to a plant for enhancing plantproductivity. In one embodiment, said microbial formulation furthercomprises a liquid carrier. In a further embodiment, said microbialformulation further comprises mixing said liquid carrier with saidmicrobial isolate. In one embodiment, said liquid carrier compriseswater and humic acid. In one embodiment, said humic acid is at aconcentration of 12% v/v (Volume of solute (ml)/Volume of solution(ml)). In one embodiment, said liquid carrier has a pH of 7.0. In oneembodiment, said microbial isolate concentration in the liquid carrierranges from 10¹⁰-10¹⁷ microbes per milliliter of liquid formulation. Inone embodiment, said applying is selected from the group consisting ofseed dipping, root dipping, seedling root dip, soil drench, pipetting,irrigating, spraying, foliar spraying, spraying at the base of theplants, and the like. In one embodiment, said plant is selected from thegroup consisting of a vegetable plant, a legume plant, a cereal plant, afodder plant, a grass plant, a fiber plant, an oil seed plant, a fieldpant, a garden plant, a green-house plant, and a house plant. In oneembodiment, said plant is selected from the group consisting of a tomatoplant, an eggplant plant, an okra plant, a squash plant, a zucchiniplant, a bean plant, a pea plant, a soybean plant, a rice plant, a cornplant, a sorghum plant, an alfalfa plant, a grass plant, a turf grassplant, a clover plant, a cotton plant, and a peanut plant. In oneembodiment, said enhancing plant productivity is increasing anagriculturally desirable trait. In one embodiment, said agriculturallydesirable trait is selected from the group consisting of percentage ofseed germination, quality of seed germination, height of the plant,width of plant, equivalent leaf area, shoot length, root length, legumenodulation, number of legume nodules, grain yield, fruit yield, shootweight, root weight, biomass, altered time for flowering, altered timefor fruit formation, decreased disease incidence, and increased diseaseresistance. In one embodiment, said agriculturally desirable trait isevaluated at 30-60 days after sowing.

The present invention provides exemplary isolated bacterial strainsselected from the group consisting of an Ensifer meliloti FD, Rhizobiumtrifolii FD, Azorhizobium caulinodans KN, Rhizobium sp. RLG1,Azorhizobium sp. RLG2, Azorhizobium sp. RLG3, Rhizobium sp. RLG4,Rhizobium sp. RLG5, Rhizobium sp. RLG6, Azorhizobium sp. RLG7, Rhizobiumsp. RLG8, Azorhizobium sp. RLG9, Rhizobium sp. RLG10, and Rhizobium sp.RLG 11 having accession number N.R.R.L. B-50215. In one embodiment atleast two of said isolated bacterial strains are provided together in amixture. In one embodiment at least fourteen of said isolated bacterialstrains are provided together in a mixture.

The present invention provides an exemplary mixture of bacterialisolates having accession number N.R.R.L. B-50215.

The present invention provides exemplary isolated fungal strainsselected from the group consisting of a Trichoderma vireni 3107,Trichoderma viride LK, Trichoderma viride 3116, Trichoderma harzianum3147, Trichoderma harzianum G, Trichoderma harzianum LK, and Trichodermalongibrachiatum 3108 fungal strain having accession number N.R.R.L.50216. In one embodiment at least at least two of said isolated fungalstrains are provided together in a mixture. In one embodiment at leastseven of said isolated fungal strains are provided together in amixture.

The present invention provides an exemplary mixture of fungal isolateshaving accession number N.R.R.L. 50216.

The present invention provides exemplary microbial formulation, whereinsaid formulation consists of a nitrogen fixing bacteria isolate, aphosphate solubilizing microbe isolate, a Rhizobacteria isolate, and abiocontrol microbe isolate. In one embodiment said microbe is selectedfrom the group consisting of a bacteria and a fungus. In one embodimentsaid biocontrol microbe is selected from the group consistingTrichoderma viride 3116, Trichoderma vixens 3107, Trichoderma harzianum3147, Trichoderma harzianum LK, Trichoderma harzianum G, and Trichodermalongibrachiatum 3108 having accession number N.R.R.L. 50216. In oneembodiment said bacteria isolate is selected from the group consistingof Ensifer meliloti FD, Rhizobium trifolii FD, Azorhizobium caulinodansKN, Rhizobium sp. RLG 1, Azorhizobium sp. RLG2, Azorhizobium sp. RLG3,Rhizobium sp. RLG4, Rhizobium sp. RLG5, Rhizobium sp. RLG6, Azorhizobiumsp. RLG7, Rhizobium sp. RLG8, Azorhizobium sp. RLG9, Rhizobium sp.RLG10, and Rhizobium sp. RLG11 having accession number N.R.R.L. B-50215.

The present invention provides an exemplary microbial formulation,wherein said formulation comprises a mixture selected from the groupconsisting of a bacterial mixture having accession number N.R.R.L.B-50215 and a fungal mixture having accession number N.R.R.L. 50216.

The present invention provides an exemplary microbial formulation,wherein said formulation is a mixture of bacteria isolates selected fromthe group consisting of Ensifer meliloti FD, Rhizobium trifolii FD,Azorhizobium caulinodans KN, Rhizobium sp. RLG1, Azorhizobium sp. RLG2,Azorhizobium sp. RLG3, Rhizobium sp. RLG4, Rhizobium sp. RLG5, Rhizobiumsp. RLG6, Azorhizobium sp. RLG7, Rhizobium sp. RLG8, Azorhizobium sp.RLG9, Rhizobium sp. RLG10, and Rhizobium sp. RLG11 having accessionnumber N.R.R.L. B-50215 and a mixture of fungal isolates selected fromthe group consisting of Trichoderma virens 3107, Trichoderma viride LK,Trichoderma viride 3116, Trichoderma harzianum 3147, Trichodermaharzianum G, Trichoderma harzianum LK, and Trichoderma longibrachiatum3108 fungal strain having accession number N.R.R.L. B-50216. In oneembodiment the formulation further comprises, a liquid carrier. In oneembodiment said liquid carrier comprises water and humic acid. In oneembodiment said humic acid is at a concentration of 12% volume of humicacid (ml)/volume of solution (ml) (v/v). In one embodiment said liquidcarrier has a pH of 7.0. In one embodiment said microbial isolateconcentration in the liquid carrier ranges from 10¹⁰-10¹⁷ microbes permilliliter of liquid. In one embodiment the formulation is selected fromthe group consisting of a liquid, a dried formulation, and a wettablepowder.

The present invention provides an exemplary method for enhancing plantgrowth, comprising, a) providing, i) A microbial formulation, whereinsaid formulation comprises a mixture selected from the group consistingof a bacterial mixture having accession number N.R.R.L. B-20215 and afungal mixture having accession number N.R.R.L. B-50216, ii) a plant,and applying said microbial formulation to a plant for enhancing plantproductivity. In one embodiment, said microbial formulation furthercomprises, a liquid carrier and mixing said liquid carrier with saidmicrobial isolate. In one embodiment, said liquid carrier compriseswater and humic acid. In one embodiment, said humic acid is at aconcentration of 12 percent. In one embodiment, said liquid carrier hasa pH of 7.0. In one embodiment, said microbial isolate concentration inthe liquid carrier ranges from 10¹⁰-10¹⁷ microbes per milliliter ofliquid formulation. In one embodiment, said applying is selected fromthe group consisting of seed dipping, pipetting, irrigating, spraying,and foliar spraying. In one embodiment, said plant is selected from thegroup consisting of a vegetable plant, a legume plant, a cereal plant, afodder plant, a grass plant, a fiber plant, an oil seed plant, a fieldpant, a garden plant, a greenhouse plant, and a house plant. In oneembodiment, said plant is selected from the group consisting of atomato, eggplant, okra, squash, zucchini, bean, pea, soybean, rice,corn, sorghum, alfalfa, Bermuda grass, clover, cotton, and peanut. Inone embodiment, said enhancing plant productivity is increasing anagriculturally desirable trait. In one embodiment, said methodagriculturally desirable trait is selected from the group consisting ofseed germination, height of the plant, leaf area, shoot length, rootlength, legume nodulation, grain yield, fruit yield, shoot weight, rootweight, biomass, altered time for flowering, altered time for fruitformation, decreased disease incidence, and increased diseaseresistance.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an exemplary preliminary greenhouse experimentdemonstrating garden pea plants treated with F1 formulation compared toa control plant (no F1). F1=Sumagro 1.

FIG. 2 shows an exemplary greenhouse experiment demonstrating garden peaplants treated with F1=Sumagro 1; F2=Sumagro 2; NG=Nutragro; andC=control treatment (Experiment 1).

FIG. 3 shows an exemplary greenhouse experiment demonstrating tomatoplants treated with F1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro &Nutragro; and C=control treatment (Experiment 1).

FIG. 4 shows an exemplary greenhouse experiment demonstrating tomatoplants, after two months of growth, treated with F1=Sumagro 1;F2=Sumagro 2; HG & NG=Humagro & Nutragro; and C=control treatment(Experiment 1).

FIG. 5 shows an exemplary greenhouse experiment demonstrating purplehull pea plants treated with F1=Sumagro 1; F2=Sumagro 2; NG=Nutragro;and C=control treatment (Experiment 1).

FIG. 6 shows an exemplary greenhouse experiment demonstrating soybeanplants treated with F1=Sumagro 1; F2=Sumagro 2; NG=Nutragro; andC=control treatment (Experiment 1).

FIG. 7 shows an exemplary greenhouse experiment demonstrating WonderBush bean plants treated with F1=Sumagro 1; F2=Sumagro 2; NG=Humagro &Nutragro; and C=control treatment (Experiment 1).

FIG. 8 shows an exemplary greenhouse experiment using Wonder Bush beanplants treated with F2=Sumagro 2; and NG=Nutragro (Experiment 1).

FIG. 9 shows an exemplary greenhouse experiment demonstrating squashplants treated with F1=Sumagro 1; F2=Sumagro 2; NG=Nutragro; andC=control treatment (Experiment 1).

FIG. 10 shows an exemplary greenhouse experiment demonstrating squashplants treated with F1=Sumagro 1; F2=Sumagro 2; NG=Nutragro; and HGHumagro (Experiment 1).

FIG. 11 shows an exemplary greenhouse experiment demonstrating tomatoplants treated with F1=Sumagro 1; F2=Sumagro 2; HG=Humagro; HG &NG=Humagro & Nutragro; and C=control treatment (Experiment 2).

FIG. 12 shows an exemplary greenhouse experiment demonstrating Eggplant(brinjal) plants treated with F1=Sumagro 1; F2=Sumagro 2; HG=Humagro; HG& NG=Humagro & Nutragro; and C=control treatment (Experiment 2).

FIG. 13 shows an exemplary greenhouse experiment demonstrating okraplants treated with F1=Sumagro 1; F2=Sumagro 2; HG=Humagro; HG &NG=Humagro & Nutragro; and C=control treatment (Experiment 2).

FIG. 14 shows an exemplary greenhouse experiment demonstrating riceplants treated with F1=Sumagro 1; F2=Sumagro 2; HG=Humagro; HG &NG=Humagro & Nutragro; and C=control treatment (Experiment 2).

FIG. 15 shows exemplary root nodules from Garden Bean plants (Rhizobialnoculum comparisons) A) Plants treated with F3 formulation (Rhizobialinoculum); B) Plants treated with F4 formulation, Note that F4formulation consisting of Trichoderma fungi only (which are non-nitrogenfixers) induced diverse type of nodule formation as shown in A byindigenous nitrogen-fixing bacteria on the roots of Garden Beans; C)bean plants grown in sterile soil treated with F2 showing modest levelsof nodulation as compared; D) Plants grown in unsterile soil treatedwith F2; to E) bean plant roots from bean plants grown in sterile soilwithout F2 treatments. Plants were treated with a formulation (oruntreated for controls) of the present invention according to methodsdescribed in Example 1.

FIG. 16 shows an exemplary comparison of corn plant biomass from a fieldtrial. F1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro & Nutragro; andC=control treatments.

FIG. 17 shows an exemplary okra fruit weight from a field trial.F1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro & Nutragro; and C=controltreatments.

FIG. 18 shows an exemplary peanut biomass from a field trial. F1=Sumagro1; F2=Sumagro 2; HG & NG=Humagro & Nutragro; and C=control treatments.

FIG. 19 shows an exemplary peanut plant yield from a field trial.F1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro & Nutragro; and C=controltreatments.

FIG. 20 shows an exemplary rice plant yield from a field trialF1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro & Nutragro; and C=controltreatments.

FIG. 21 shows an exemplary comparison of rice plant height A) from afield trial, and grown under Greenhouse Evaluation conditions B) plantheight and C) yield of rice grown in the presence of polymicrobialformulations F1 and F2 as compared to a control with no formulationadded. F1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro & Nutragro; andC=control treatments.

FIG. 22 shows an exemplary comparison of soybean plant A) biomass from afield trial and grown under Greenhouse Evaluation conditions plantheight B) and yield C) of soybean plants grown in the presence ofpolymicrobial formulations F1 and F2 as compared to a control with noformulation added. F1=Sumagro 1; F2=Sumagro 2; HG & NG=Humagro &Nutragro; and C=control treatments.

FIG. 23 shows an exemplary comparison of A) tomato plant height from afield trial and grown under Greenhouse Evaluation conditions plantheight B) and yield C) of tomato grown in the presence of polymicrobialformulations F1 and F2 as compared to a control with no formulationadded under Greenhouse Evaluation conditions. F1=Sumagro 1; F2=Sumagro2; NG=Nutragro; and C=control treatments.

FIG. 24 shows an exemplary comparison of Wonder bush beans grown underGreenhouse Evaluation conditions plant height A) and yield B) grown inthe presence of polymicrobial formulations F1 and F2 as compared to acontrol with no formulation added

FIG. 25 shows an exemplary comparison of growth observed in a mixture ofpotted grass plants (commercial forage seed mixture Tecomate MonsterMix) treated with Mineral solution (MM), HG (F2 prepared in a carrier ofHG; F2+HG), NF2 (F2 prepared in a carrier of mineral solution (MM) inplace of HG). HG=Humagro; F2=Sumagro 2.

FIG. 26 shows an exemplary dramatic increase in growth of clover plantsA) grown with F2 treatments as compared to B) control plants withouttreatments.

FIG. 27 shows an exemplary growth enhancing effect of formulations ofthe present inventions on several types of switch grass plant varietiesA) Carthage, B) Cave-in-Rock C) Forestburg and D) Dacotah (Dakota).

FIG. 28 shows an exemplary root nodule formation in pea plants treatedwith formulations consisting of either, A) bacterial strains (F3) and B)Trichoderma strains (F4).

FIG. 29 shows an exemplary biological control effect of fungal isolatesof the present inventions on a pathogenic fungus A) A—Alternariaalternata (plant pathogen sometimes called a Tomato leaf spot pathogen)grown in the presence of fungal isolates of the present inventions:TH—Trichoderma harzianum (showing Bio-control of fungus); TV—T. viride(showing Bio-control of fungus); TL—T. longibrachiatum (showingBio-control of fungus) and B) A—Alternaria alternata (plant pathogensometimes called a Tomato leaf spot pathogen); C—Curvularia sp. (Tomatoleaf spot pathogen); and F—Fusarium solani (Tomato pathogen) grown nextto B5—Antagonistic Bacterial strain Pseudomonas fluorescens isolate ofthe present inventions.

FIG. 30 shows an exemplary comparison of formulations demonstratingbiocontrol against Powdery mildew: A) soybean plants infected by Powderymildew showing that an F2 treated Soybean plant (4) is free from Powderymildew infection unlike the plants treated merely with conventionalfertilizer (NPK) (plants 1, 2 and 3) and squash plants exposed topowdery mildew in a greenhouse B) with F2 treatments and C) without F2treatments.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases as used herein are defined below:

The use of the article “a” or “an” is intended to include one or more.

As used herein, terms defined in the singular are intended to includethose terms defined in the plural and vice versa.

As used herein, “formulation” in reference to a composition of thepresent invention refers to a product, wherein “formulating” is theprocess of using a formula, such as a recipe, for a product, i.e. theingredients, the quantities ingredients that were or would be added, thesequence of adding an ingredient, and the processing steps that were orwill be taken to provide the product. A formulation may be in any form,such as a liquid, solid, i.e. dried formulation, wettable powder, and insome embodiments, applied with a carrier.

As used herein, the term “applying” in reference to a formulation of thepresent invention refers to any means for treating seeds, soil, andplants with formulations of the present inventions, for example, seeddipping, soil drench, pipetting onto soil, pipetting onto plants,irrigating plants with liquids comprising formulations of the presentinventions, spraying formulations of the present inventions, i.e. foliarspraying, and the like. “Applying to a plant” refers to any means fortreating a plant with a formulation of the present inventions, forexample, adding the formulation to the soil at any time prior to, incombination with, or after planting anyone of a seed, seedling, orgrowing plant.

As used herein, the term “seed dipping” refers to application of aformulation of the present invention directly to a seed, such as soakinga seed for few seconds, minutes, or hours in a liquid formulation of thepresent inventions. Seed dipping may also refer to application of adried microbial formulation of the present inventions.

As used herein, the term “soil drench” refers a to applying a liquid tosoil.

As used herein, “carrier” in reference to formulations of the presentinventions, refers to a substance, either synthetic or natural, fortransporting an active ingredient, such as a microbe of the presentinventions, onto a plant, a seed, soil, etc, examples of a carrierinclude, humic acid, a mineral, a botanical, and the like.

As used herein, “agent” in reference to an ingredient of a formulationof the present inventions, refers to a substance that causes a change,such as a chemical agent or a substance that protects an activeingredient of a formulation, for example, an ultraviolet light resistantagent, etc.

As used herein, “dispersing agent” refers to a material that will causemicrocapsules or particles to separate uniformly throughout a solid,liquid, or gas. Alternatively, a “dispersing agent” refers to a materialthat will cause a dispersion of microbes, microcapsules or particlesinto the environment, for example, a dispersing agent will allow anactive ingredient to be dispelled from a microcapsule, a dispersingagent will allow an active ingredient to be dispersed into theenvironment.

As used herein, “inert” in reference to an ingredient of theformulations of the present inventions, refers to a material that is notreadily reactive with other materials, such as ingredients or host orenvironmental materials, such that an inert ingredient forms few or nochemical compounds.

As used herein, “fill material” in reference to an agent or aningredient of the present inventions, refers to a substance for“filling” in the spaces of a capsule of the present inventions, suchthat the active ingredient may be referred to as a fill material inaddition to any fill material desired for incorporation in a formulationof the present inventions, for example, gelatin, hydrogel, etc. A fillmaterial may be inert, may comprise a controlled release agent, may be areleasable fill material, and may be an active agent, and a combinationthereof, in the formulations of the present inventions.

As used herein, the term “stabilizer” refers to a substance capable ofimparting resistance against physical or chemical deterioration ordecomposition, for example, a fill material or fill stabilizer and ashell stabilizer, see, as an example, United States Patent ApplicationNo. 20030202999, herein incorporated by reference in its entirety.

As used herein, “agronomically acceptable salts” refers to mineral saltsthat do not induce negative effects on agricultural crops when usedproperly. They include, metal salts such as sodium, potassium, calciumand magnesium salts, ammonium salts such as isopropyl ammonium salts andtrialkylsulfonium salts such as triethylsulfonium salts.

As used herein, “phytohormones” refers to a plant hormone including anyof the hormones produced naturally in plants and that are active inminute amounts in controlling growth and other functions at a siteremote from the place of production. The three principal types areauxins, cytokinins and gibberellins.

As used herein, the term “plant” is used in its broadest sense. Itincludes, but is not limited to, any species of grass (e.g. turf grass),sedge, rush, ornamental or decorative, crop or cereal, fodder or forage,fruit or vegetable, fruit plant or vegetable plant, woody, flower ortree. It is not meant to limit a plant to any particular structure. Suchstructures include, but are not limited to, stomata, a seed, a tiller, asprig, a stolon, a plug, a rhizome, a shoot, a stem, a leaf, a flowerpetal, a fruit, etc.

As used herein, the terms “crop” and “crop plant” are used herein itsbroadest sense. The term includes, but is not limited to, any species ofplant or alga edible by humans or used as a feed for animals or fish ormarine animals, or consumed by humans, or used by humans, or viewed byhumans (flowers) or any plant or alga used in industry or commerce oreducation, such as vegetable crop plants, fruit crop plants, fodder cropplants, fiber crop plants, and turf grass plants.

As used herein, the terms “leaf” and “leaves” refer to a usually flat,green structure of a plant where photosynthesis and transpiration takeplace and attached to a stem or branch.

As used herein, “stem” refers to a main ascending axis of a plant.

As used herein, “seed” refers to a ripened ovule, consisting of theembryo and a casing.

As used herein, “pathogen” refers a biological agent that causes adisease state (e.g., infection, anthracnose, etc.) in a host.“Pathogens” include, but are not limited to, viruses, bacteria, archaea,fungi, protozoans, mycoplasma, parasitic organisms and insects.

As used herein, the terms “bacteria” and “bacterium” refer to allprokaryotic organisms, including those within all of the phyla in theKingdom Prokaryotae. It is intended that the term encompasses allmicroorganisms considered to be bacteria, including Azotobacter,Azospirillum, Azorhizobium, Pseudomonas, Bacillus, Rhizobium,Mycoplasma, et cetera. All forms of bacteria are included within thisdefinition including cocci, bacilli, spirochetes, spirilla, vibrios,spheroplasts, protoplasts, etc. Also included within this term areprokaryotic organisms that are gram negative or gram positive. “Gramnegative” and “gram positive” refer to staining patterns with theGram-staining process that is well known in the art. (See, e.g.,Beveridge, 2007, Sampling and staining for light microscopy, pages19-33. In C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T.M. Schmidt, and L. R. Snyder (eds.). Methods for General and MolecularMicrobiology, Am. Soc. Microbiol., Washington, District of Columbia.“Gram positive bacteria” are bacteria which retain the primary dye usedin the Gram stain, causing the stained cells to appear dark blue topurple under the microscope. “Gram negative bacteria” do not retain theprimary dye (crystal violet) used in the Gram stain, but are stained redby the counter stain (safranin). Thus, Gram negative bacteria appearred.

As used herein, “microorganism” refers to any species or type ofmicroorganism, including but not limited to, bacteria, archaea, fungi,protozoans, mycoplasma, and parasitic organisms.

As used herein, “fungi” is used in reference to eukaryotic organismssuch as the molds and yeasts, including dimorphic fungi, fungi found insoil, and any fungi found growing on a plant.

As used herein, “cfu” refers to a colony forming unit.

As used herein, “enhancement” refers to increasing a characteristic,such as growth, grain yield.

As used herein, “fertilizer” refers to any organic material or inorganicmaterial of natural or synthetic origin which is added to soil toprovide nutrients, including all three elements of nitrogen, phosphorus,and potassium, necessary to sustain plant growth.

As used herein, “humic molecule” as used herein means a carbon moleculewith open and available hydrogen and oxygen bonding sites and exchangecapacity.

As used herein, “humate-based” refers to include but are not limited tosugars (including glucose, fructose, and molasses), plasma, manure tea(for example, colored water that manure has been steeped in), peatextracts, compost extracts, coal extracts, leonardite extracts, kelp orextracts thereof, and other humic matrices known in the art that containhumic molecules that are rich in macronutrients, nitrogen, phosphorusand/or potassium. The matrix may also contain growth-stimulatingcompounds such as a blend of botanic/carbohydrates, growth factors,amino acids and micro-nutrients including calcium, boron, copper,molybdemum, manganese, magnesium, iron, sulfur and zinc as needed.

As used herein, “herbicide” refers to any substance, either synthetic ornatural, used to kill a plant or inhibit plant growth. Typically anherbicide is intended to kill a weed while leaving the desired plant,such as a crop plant, alive.

As used herein, the term “weed” refers to any plant a plant grower, suchas a farmer, landscaper, and the like, would like to eliminate that isgrowing in a container, such as a pot, or in a field, for example, aweed is a grass plant growing in a beet field.

As used herein, “pesticide” or “biocide” refers to a substance ormixture of substances intended for destroying, killing, repelling,mitigating the life of any pest. Pests can be insects, mice and otheranimals, unwanted plants (weeds), fungi, algae, or microorganisms likebacteria and viruses. The term pesticide also applies to herbicides,fungicides, and various other substances used to control pests.

As used herein, “pest” refers to a living organism that occur where theyare not wanted or that cause damage to crops or humans or other animals,examples include insects; mice and other animals; unwanted plants (suchas weeds); fungi; algae, and microorganisms such as bacteria andviruses.

As used herein, “plant growth regulator” or “PGR” refer to a chemicalthat affects plant growth and/or development.

As used herein, “diverse” refers to a group of different microbes, suchas a group comprising a gram+bacterium, gram−bacterium, a motilemicrobe, a nonmotile microbe, a root nodule microbe, a soil microbe, arhizosphere microbe, a fungus, and the like.

As used herein, “multifunctional” in reference to a formulation refersto a formulation providing at least 2 functions, for example, a healthyplant, a net result healthy plants, nutrients, higher productivity,faster growth, then microbes also synergistic effects.

As used herein, “functionality” refers to increasing plant growthproductivity, inducing pest resistance, nutrient cycling.

As used herein, “broad spectrum” in reference to beneficial resultsrefers to benefits to a combination of leguminous and nonleguminous,vegetable crops and other plants and described herein.

As used herein, “broad spectrum” in reference to plants refers to anytype of plant.

As used herein, “safe” in reference to environmental activity refers toa condition of exposure under which there is a practical certainty thatno harm will result to the ecosystem, such a the surrounding ground,air, and water, including ground water, surface water, drainage waterand any bodies of water into where drainage water flow.

As used herein, “ecological impact” refers to an effect that aman-caused or natural activity has on living organisms and theirnon-living (abiotic) environment.

As used herein, “ecological sustainability” or “environmentalsustainability” refers to current methods of ecosystem maintenance,including components and functions, in order to provide safe and healthyecosystems for future generations of plants, fish, reptiles, mammals,and microbial communities.

As used herein, “ecology” refers to a relationship of living things toone another and their environment, or the study of such relationships.

As used herein, “ecosystem” refers to an interacting system of abiological community, including but not limited to plants, fish,reptiles, mammals, and microbial communities, and its non-livingenvironmental surroundings, such as soil, water, and air.

As used herein, “road” refers to any highway, road, street, avenue,lane, private way, and similarly paved, gravel or dirt thoroughfare forany type of vehicle, airplane, train, bicycle, animal and human.

As used herein, “tilth” in reference to soil refers to a physicalcondition of the soil as related to tillage, seedbed preparation,seedling emergence, and root penetration.

As used herein, “NRRL” or “N.R.R.L.” in reference to a biologicaldepository for microorganisms recognized under the Budapest Treaty,refers to the “Agricultural Research Service Culture Collection (ARS)National Center for Agricultural Utilization Research, in Peoria, Ill.United States of America.”

As used herein, “accession number” in reference to “having an accessionnumber” refers to a number assigned to a cultured isolate upondeposition to a recognized depository of patent strains, for example,Paenibacillus peoriae, strain NRRL BD-62, where BD-62 is an accessionnumber.

As used herein, “isolate” refers to a pure microbial culture separatedfrom its natural origin, such an isolate obtained by culturing a singlemicrobial colony. In other words, an isolated bacterial strain, forexample, an Ensifer meliloti FD, Rhizobium trifolii FD, Azorhizobiumcaulinodans KN, Rhizobium sp. RLG1, Azorhizobium sp. RLG2, Azorhizobiumsp. RLG3, Rhizobium sp. RLG4, Rhizobium sp. RLG5, Rhizobium sp. RLG6,Azorhizobium sp. RLG7, Rhizobium sp. RLG8, Azorhizobium sp. RLG9,Rhizobium sp. RLG10, Rhizobium sp. RLG11, etc. and an isolated fungalstrain, for example, Trichoderma viride 3116, Trichoderma virens 3107,Trichoderma harzianum 3147, Trichoderma harzianum LK, Trichodermaharzianum G, Trichoderma longibrachiatum 3108, etc.

As used herein, “mixture” refers to a combination of two or moresubstances that are not chemically united. Mixtures may be naturalmixtures and man-made mixtures, such as mixtures of the presentinventions, for example, a mixture of microbial isolates. A mixture maybe physically separated into individual substance, such as in thepresent inventions wherein a microbial isolate may be re-isolated from amixture of isolates.

As used herein, “enhancing plant productivity” refers to any aspect of aplant altered for a “desired benefit,” such as increasing anagriculturally desirable trait. “Desired benefit” also refers to anyeffect on a plant to confer a benefit to humans and animals,

As used herein, “agriculturally desirable trait” refers to anyqualitative and quantitative agricultural trait, such as crop yield,biomass, resistance to pathogens, resistance to pests, resistance toenvironmental changes, for example, drought, etc. In other words, adesirable trait is any characteristic worth obtaining.

GENERAL DESCRIPTION

The present invention relates to eco-friendly compositions and methodsfor providing plant growth enhancing formulations comprising mixtures ofmicrobial isolates. In particular, numerous bacterial and fungal strainswere isolated from a variety of soil types, from rhizospheres and fromroot nodules of leguminous plants, in designed combinations, forproviding plant growth and plant productivity enhancing formulations.These specifically designed polymicrobial formulations would furtherprovide protection against plant pathogens lowering the need fornitrogen containing fertilizers, solubilize minerals, protect plantsagainst pathogens, and make available to the plant valuable nutrients,such as phosphate, thus reducing and eliminating the need for usingchemical pesticides and chemical fertilizers. Thus it would greatlyenhance the environment to use polymicrobial-derived products, forincreasing plant growth and productivity and for reducing the neednitrogen fertilizers and pesticides.

The inventors contemplate numerous advantages of using polymicrobialformulations of the present inventions. In particular, unlike otherformulations, the inventors specifically designed new formulations forenhancing plant growth and productivity by specifically choosingcombinations of microbes demonstrating specific microbial traits. Thebenefits conferred by these formulations include symbiotic andnon-symbiotic nitrogen fixation, suppression of disease causing organismand induction of systemic resistance in plants, helping enhance nutrientuptake by solubilizing plant nutrients such as phosphorous, productionof plant hormones and micronutrients that stimulate growth andproductivity and better tolerance to environmental stress.

In addition to formulations providing at least one benefit to plants,such as a biofertilizer, for protection against plant pathogens(biocontrol), growth nutrients, etc., the inventors also provided oneformulation for providing at least 4 specific types of benefits toplants: nitrogen fixation (legume and nonlegume); biocontrol of plantpathogens, phosphate solubilization; and plant growth promotersubstances. In other words, certain formulations of the presentinventions provided a combination of biofertilizer, biocontrol, andplant growth promoter substances. These four basic benefits are providedby a combination of microbes where nutrients are provided directly tothe plants by the microorganisms or indirectly by providing benefits toplants by inducing endogenous microbial communities to provide benefits,such as growth and productivity. Thus, unlike the microbial formulationscurrently available, the inventors contemplated a polymicrobialformulation comprising at least four groups of organisms; nitrogenfixing (including both symbiotic and nonsymbiotic nitrogen fixationmicrobes), biocontrol microbes (bacteria and fungal), PhosphorusSolubilizing Microorganisms (PSM), and Plant Growth PromotingRhizobacteria (PGPR) for providing plant growth promoter substances.Further, the inventor included microbes with additional capabilities,for example, microbes for inducing nodule formation in roots.

Therefore, inventors designed specific polymicrobial formulations forproviding multiple benefits to plants and their crops by combiningtraits of specific microbes. These polymicrobial formulations weredesigned to provide a specific combination of benefits to plants, humansand their environment. These combined benefits are not found inindividual microbial isolates used as inoculums. Examples of traits fromindividual microbe Genus or species for providing specific benefits toplants.

During the course of development and testing of the present inventionsin field trials, the inventors found that using formulations of thepresent inventions, in particular Sumagro-2, the amount of traditionalchemical fertilizer application was reduced at least 50% while retainingdesired agricultural traits. In Greenhouse trials the formulations wereprimarily used without chemical or artificial fertilizer or other typeof chemical supplementation. Thus the inventors' contemplate the use ofthese formulations in combination with traditional chemical use wherethe amount of traditional chemical application is substantially reduced.In one embodiment, the use of chemicals is reduced at least 25%. Inanother embodiment the use of chemicals is reduced at least 30%. Inanother embodiment the use of chemicals is reduced at least 40%. Inanother embodiment the use of chemicals is reduced at least 50%. Inanother embodiment the use of chemicals is reduced at least 60%. Inanother embodiment the use of chemicals is reduced at least 75%. Inanother embodiment the use of chemicals is reduced at least 80%. Inanother embodiment the use of chemicals is reduced at least 90%. In apreferred embodiment, the use of formulations of the present inventionscompletely replaces the use of chemical treatments in the home,greenhouse and field.

Even further, the inventors contemplated formulations of the presentinventions such that one formulation would provide at least fourbenefits listed above and would also provide benefits to a broadspectrum of plants, including but not limited to legumes and nonlegumes,cereals and grains, vegetables and fruits, fiber producing plants andgrass plants. The inventors' further contemplate the use of theformulations on additional types of plants including but not limited totrees. These formulations would provide benefits that include but notlimited to agricultural traits, such as increasing crop yield andbiomass. The inventors further contemplate the use of their formulationsas prophylactic treatments for biocontrol of pathogens, includingbacteria, fungus, nematodes and insects.

Soil microbial populations often contribute to the growth and health ofplants including but not limited to crop plants, landscaping plants,garden plants, greenhouse plants, indoor plants, et cetera. Microbeswithin these populations perform a variety of functions, such asconverting atmospheric nitrogen, which plants cannot use, into ammoniaor other useful nitrogenous compounds that many plants can use. Forexample, nitrogen conversion (wherein “conversion” is also referred toas “fixation”) takes place in small nodules on the roots of legumes,such as pea plants, bean plants, soybean plants, clover plant plants, etcetera. Thus in one embodiment, the inventors contemplated isolatingmicrobes for increasing nitrogen fixation in plants. In one embodiment,the inventors contemplated using microbes for increasing nitrogenfixation. In one embodiment, the inventors contemplated using microbialmixtures for increasing legume nodule formation. In a furtherembodiment, the inventors contemplated using microbial mixtures forinducing legume nodule formation. In some embodiments, the inventorscontemplated the isolation and use of Plant Growth PromotingRhizobacteria (PGPR), which are generally root-colonizing bacteria.

Microbes also oxidize chemicals and assist plants in absorbing nutrientsand trace elements, such as phosphates, iron, cobalt, manganese, andmolybdenum, from soil in addition to decomposing plant and animalorganic matter into simpler organic products that plants can absorb anduse to sustain their growth. In particular, cultivatable soil isfrequently alkaline in nature containing calcium or magnesium withlittle available phosphorus. Due to a typically higher concentration ofcalcium, whenever phosphatic fertilizers are applied in such soil, alarge quantity of applied phosphate gets fixed as Tri-Calcium Phosphatewhich is water insoluble and hence becomes unavailable to the plant.Conversely, when soil is acidic, iron or aluminum salts will form withapplied phosphate containing fertilizers. Fortunately, certain soilmicroorganisms have inherent capacity to dissolve part of the fixedphosphorus (salts) and make it available to the crop by secretingcertain organic acids. These types of organisms are called PhosphorusSolubilizing Microorganisms (PSM). Soil bacteria and fungi comprise thegreatest percentages of phosphate Solubilizing microorganisms, known asPSM or Phosphate Solubilizing Bacteria (PSB) and Phosphate SolubilizingFungi (PSF). These microorganisms are capable of Solubilizing insolublecompounds and release phosphorus to soil solution. Soil microbes furtherassist in forming and maintaining arable soils rich in complex organicmaterials through which roots easily grow and absorb water andnutrients.

Thus the inventors also contemplated the isolation of microbes forenhancing plant growth and productivity, in particular bacteria, foroxidizing chemicals and assisting plants to increase absorption ofnutrients and trace elements.

However microbial populations and individual microbe species may also bedetrimental to plant health. Deleterious rhizobacteria (DRB) arepredominantly saprophytic bacteria that aggressively colonize plantseeds, roots and rhizospheres and readily metabolize organic substancesreleased by plant tissues. Numerous types of plant diseases, below andabove-ground, are caused by both individual species and groups ofbacteria found in the soil. Furthermore, harmful molds and other soilfungi are responsible for many serious root diseases and above-grounddiseases of plants. Thus, major economic crops frequently are damaged bysoil-borne fungi and bacteria, for example, root rots, collar rots,wilts, seed decay, seedling blights, fruit rots, root browning,damping-off, etc., take a heavy economic toll each year. Many of theseplant diseases caused by soil-borne plant pathogens (where pathogensrefer to any disease-causing organism) are difficult to control byconventional procedures, for example, by using synthetic chemicalpesticides.

Even further, the inventors further contemplate isolating and usingmicrobial isolates and mixtures of isolates for biocontrol ofphytopathogenic organisms. Biocontrol agents are also useful in a methodof enhancing plant growth that involves applying them to plants, plantsseeds, or soil surrounding plants under conditions effective to enhancegrowth in the plants or plants produced from the plant seeds. Evenfurther, the formulations are contemplated for use in insect biocontroltreatments and programs.

Numerous attempts have been made by others to provide microbial-basedproducts for beneficial plant growth, however few, if any, effectivecommercially viable products are available. The lack of highly effectivemicrobial based products is not consistent with the numerouspublications showing that plant growth can sometimes be affectedbeneficially when inoculated with one specific isolated microbialspecies, such as a bacteria or fungi, for example, a Pseudomonasfluorescens NBRI 1303 (ATCC 55939) isolate was shown to be effective insuppressing plant pathogens, including Fusarium oxysporum f. sp. ciceri,Rhizoctonia bataticola and Pythium sp. in chickpeas (U.S. Pat. No.6,495,362; herein incorporated by reference) while other bacteria weredisclosed as single bacterium isolates (for example, U.S. Pat. No.6,896,883; herein incorporated by reference), in addition tocombinations of multiple strains from one Genus, (for example, U.S. Pat.No. 6,194,193; herein incorporated by reference) and multiple microbes(for example, Publication Number: WO/2005/077861; herein incorporated byreference). However none of these publications show a singleformulations providing the multiple benefits for a broad spectrum ofcrops as described for formulations of the present inventions.

Other microbes for providing disease resistance were also reported, (forexample, U.S. Pat. No. 6,495,362, U.S. Pat. No. 6,280,719; hereinincorporated by reference). A T. harzianum T22 fungal strain wasreported to enhance root development from field-grown corn and soybeanplants and improve survival of pepper plants ((Trichoderma spp.,including T. harzianum, T. viride, T. koningii, T. hamatum and otherspp. Deuteromycetes, Moniliales (asexual classification system) (Harman,Cornell Community Conference on Biological Control, Apr. 11-13, 1996;www.nysaes.cornell.edu/ent/biocontrol/pathogens/trichoderma.html; hereinincorporated by reference) in addition to findings that “Seed treatmentwith Trichoderma harzianum strain T22, which results in colonization ofplant roots but little or no colonization of shoots or leaves, hadsubstantial effects on growth of and disease expression in maize inbredline Mo17. Shoots and roots of 10-day-old seedlings grown in a sandyloam field soil were larger (roots were nearly twice as long) in thepresence of T22 than in its absence . . . . Plants grown fromT22-treated seed had reduced symptoms of anthracnose followinginoculation of leaves with Colletotrichum graminicola, which indicatesthat root colonization by T22 induces systemic resistance in maize.”(Harman, et al., The American Phytopathological Society, Vol. 94, No. 2,2004, 147-153, herein incorporated by reference). Further, “specificstrains of fungi in the genus Trichoderma (T.) colonize and penetrateplant root tissues and initiate a series of morphological andbiochemical changes in the plant, considered to be part of the plantdefense response, which in the end leads to induced systemic resistance(ISR) in the entire plant. The capability of T. harzianum to promoteincreased growth response was verified both in greenhouse experimentsand in the hydroponic system.” (Chet, et al., Plant Biocontrol byTrichoderma spp., Weizmann Institute,www.weizmann.ac.il/Biological_Chemistry/scientist/Chet/Chet.html, Lastupdated Jan. 19, 2006; herein incorporated by reference). However, thislast reference fails to refer to the strain used or type of plantdemonstrating these effects. Further, formulations of these microbialstrains would not provide the range of benefits of the polymicrobialformulations of the present inventions for a wide range of crops.

In spite of much published information on the benefit of individualmicrobes to plants (Ilungo et al 2004), there are relatively fewefficacious microbial inoculant products capable of conferring all thebeneficial effects on crop productivity (Actinovater series, HortEnterprises, Mitcom Consultancy, Nitagen, Inc., Nutragro, and TandjeEnterprises). More importantly, developing microbial formulationscontain a range of microorganisms isolated from divers rhizosphereenvironments and possessing divers functional abilities and be able toenhance production of a broad spectrum of plants has been a realchallenge. Many potentially useful bacteria never appear on thecommercial market, which may be due to inappropriate designing of theformulation, lack of efficacy under field conditions, or lack ofstability of the product. Development of a successful microbialinoculant involves several critical elements such as strain selection,selection of a carrier, mass multiplication (division and growth)appropriate construction of the formulation, and packaging andmarketing.

However, merely because one strain of microbe may be beneficial to aplant, does not mean that another strain, even of the same species, willprovide equal benefits, (for example, out of 17 rhizobacteria isolates,one Bacillus spp. out preformed the others for providing benefits towheat plants, see, Hafeez, et al., (2006) “Plant growth-promotingbacteria as biofertilizer,” Agron. Sustain. Dev. 26:143-150. [abstractonly]; herein incorporated by reference). In fact isolated strainswithin the same species may show opposite effects when used to treat aplant. One example of such dichotomy is shown by some Serratia (S.)strains, such as S. proteamaculans 1-102 and S. liquefaciens 2-68, thathave beneficial effects on legume plant growth (Chanway et al., 1989,Soil Biology and Biochemistry 21:511-517; Zhang et al., 1996, Plant andSoil 179:33-241; Bai, et al., 2002, Journal of Experimental Botany,53(373):1495-1502; all of which are herein incorporated by reference).While other Serratia strains, such as a Serratia plymuthica strain A153,may actually inhibit plant growth where it showed stronggrowth-suppressing activities against a range of broad-leaved weedsafter foliar spraying (for example, see, Weissmann, et al., 2003,BioControl, 48(6); 725-742; herein incorporated by reference).

The same types of dichotomy found with individual strains of microbesare also found in with mixtures comprising at least 2 or more strains,even when one strain was a known beneficial strain. Because mixtures ofmicrobial species may or may not be beneficial to any particular type ofplant, or a variety of plant, or to a range of plant species, eachmicrobial mixture needs to be tested on the desired types of plants,such that “desired plants” are the plants the grower intends tocultivate (grow) in order to determine whether the mixture provides anydesired benefit to a plant, where a “desired benefit” is any one ofenhancing plant growth and plant productivity, such as those describedherein, including but not limited to those demonstrated by mixtures ofthe present inventions. For one example, see, U.S. Pat. No. 6,194,193;herein incorporated by reference). To the best of the inventor'sknowledge no microbial formulation on the market is specificallydesigned to contain a comprehensive set of microbial groups withmultiple complementary functions combined with documented efficacy forsubstantially increasing productivity of such a broad spectrum ofimportant plants. A broad spectrum of plants that includes but is notlimited to cereals, vegetable, and forage crops as reported herein.

Heavy use of chemical fertilizers and pesticides that are often employedfor increasing crop productivity now result in leaching of nitrateswhich at high levels pose a health hazard to humans. Further more, whensoils become anaerobic, nitrate (NO₃) is reduced to nitrous oxide N₂O,which is over 300 times more potent than CO₂ as a greenhouse gas.Polymicrobial formulations such as those of the present inventions arecontemplated for providing a substantial decrease in the need fornitrogenous fertilizer applications to soil (by almost 50%) and furtherfor a substantial decrease in the amount of chemical pesticide use.Therefore, polymicrobial formulations, such as F2, subformulations ofF2, overlapping formulations of F2, and contemplated formulations usingisolates of the present inventions showing an increase in potency overF2, have the potential to greatly increase crop productivity with lessdependence on chemical fertilizers and pesticides. The use ofpolymicrobial formulations of the present inventions would greatlyreduce the cost of plant cultivation while alleviating negative healthand environmental consequences associated with the use of toxic chemicalcompounds. Polymicrobial formulations would also help solubilize keyplant nutrients such as phosphate and make it more available for uptakeby the plant. Moreover, products such as F2, consisting of microbes thatnaturally occur in nature, are eco-friendly, conserve soil health inincreasing the number of bacteria beneficial to crop productivity,ensure better utilization of our natural resources, and are highlycompatible with sustainable agricultural practices.

Two other important considerations by the inventors were thecost-effectiveness of the formulation the relative stability of theproduct with the organism remaining viable for at least a few months atambient temperature (Ilungo, 2004). Moreover, the microbial products onthe market with some promise of efficacy are priced too high with somecosting as much as $25.00 or more per acre. None the less, it is likelythat the research for efficacious microbial inoculants will become moreintensive in the further because of the obvious advantages with theseproducts in minimizing the input of nitrogen fertilizer and chemicalpesticides as described herein. It is likely that there would be agreater use in the future of efficacious microbial inoculantformulations in agriculture and land management strategies resulting inmore efficient crop production in a eco-friendly manner. The currenttrend is that consumers are willing to spend high amounts to supportfood products produced by such organic farming.

In one embodiment, the present invention contemplates artificialmixtures of microbial populations for use in formulations of the presentinventions for enhancing the growth of plants. Further, the formulationsprovided herein were shown to be beneficial for plant growth and plantproductivity as described herein.

I. Types of Microbes Found in Soil that were Isolated and DescribedHerein.

In general, soil bacteria may be classified as nitrogen fixing andnon-nitrogen fixing. Two major types of nitrogen fixing bacteria(diazotrophs) are known: symbiotic nitrogen-fixing bacteria asexemplified by Rhizobium, Azorhizobium, Sinorhizobium, and Ensiferspecies and free-living nitrogen-fixing bacteria such as Paenibacillus,Azospirillum, and various others. The inventors collected soil samplesthen isolated in pure culture numerous types of diazotrophicrhizobacteria, including symbiotic diazotrophs, such as Azorhizobiumcaulinodans KN, Bradyrhizobium japonicum, Rhizobium trifolii FD,Rhizobium meliloti FD, Rhizobium phaseoli and free living diazotrophs,such as Azospirillum, Acetobacter sp. LK. These bacteria were isolatedfrom two types of sources, nitrogen-fixing nodules of a variety ofleguminous species and from rhizospheres representing tropical andsubtropical soils.

Soil-borne fungal species, in addition to causing disease, may alsofunction to prevent disease. For example, “Trichoderma spp. are fungipresent in substantial numbers in nearly all agricultural soils and inother environments such as decaying wood. Among their other activities,such as inhibiting the growth of plant pathogens, they grow tropicallytoward hyphae of other fungi, coil about them in a lectin-mediatedreaction, and degrade cell walls of the target fungi. This process(mycoparastitism) limits growth and activity of plant pathogenic fungi.In addition to, or sometimes in conjunction with mycoparasitism,individual strains may produce antibiotics. However, numbers and thephysiological attributes of wild strains are not sufficient for highlyeffective control of plant diseases.” (Trichoderma for Biocontrol ofPlant Pathogens: From Basic Research to Commercialized Products, Harman,Cornell Community Conference on Biological Control, Apr. 11-13, 1996,www.nysaes.cornell.edu/ent/bcconf/talks/harman.html). “Trichodermastrains are more efficient for control of some pathogens than others,and may be largely ineffective against some fungi. The recent discoveryin several labs that some strains induce plants to “turn on” theirnative defense mechanisms offers the likelihood that these strains alsowill control pathogens other than fungi . . . . Further, plant growthpromotion: For many years, the ability of these fungi to increase therate of plant growth and development, including, especially, theirability to cause the production of more robust roots has been known.(Trichoderma spp., including T. harzianum, T. viride, T. koningii, T.hamatum and other spp. Deuteromycetes, Moniliales (asexualclassification system) (Ascomycetes, Hypocreales, usually Hypocrea spp.,are sexual anamorphs, this life stage is lacking or unknown forbiocontrol strains)” (Harman, Cornell Community Conference on BiologicalControl, Apr. 11-13, 1996,www.nysaes.cornell.edu/ent/biocontrol/pathogens/trichoderma.html), inaddition to their antifungal properties, for example, U.S. Pat. No.6,280,719; herein incorporated by reference).

Trichoderma are free living and fast growing fungi in soil and rootecosystems of many plants. Trichoderma have been demonstrated to inhibita broad spectrum of root pathogens and foliar pathogens (Harmon et al.,2004, Mathivanan et al 2000) by one or more of the following mechanism:antibiosis, antagonism, competitive exclusion, production ofphytohomones, phosphate solubilization, and serving as biochemicalelicitors of disease resistance, Furthermore, Pseudomonas andTrichoderma species which function as bio-control agents do not inhibitarbuscular mycorrhizal fungi, what are very useful in positivelyinfluencing the mineral nutrition (especially P) of the plant.

Therefore, the inventors contemplated mixtures comprising known fungalisolates. Further, the inventors' contemplated isolating novel fungalisolates. Even further, the inventors contemplated mixtures comprisingisolates of the present inventions, such as a variety of Trichodermaspecies as described herein, to expand their spectrum of antifungalactivity and began testing mixtures of fungal microbial formulations,with and without bacteria isolates. See, Examples.

Nitrogen fertilizer is the highest on-farm cost item needed for cropproduction. So, the most effective way to increase crop productivity isthrough management of nitrogen fertilizer supply to the plant. Hence,nitrogen-fixing microbes are highly beneficial in minimizing on-farmcost of crop production. The inventors contemplate that microbialformulations designed by them and containing both symbiotic andfree-living nitrogen-fixing bacteria would enhance the growth andproductivity of a broad spectrum of crops.

The inventors contemplate that microbial formulations containing acomplex mixture of microbial organisms would be able to enhance plantgrowth and productivity of a number of commercial crops of interest.Further, the inventors contemplated that in one embodiment, the use ofmicrobial mixtures of the present inventions would reduce the use of thechemical pesticides on that plant application. Preferably, the inventorscontemplated that in one embodiment, the use of microbial mixtures ofthe present inventions would eliminate the use chemical pesticides onplants. Such an approach for reducing and eliminating the use of harmfulchemicals would be ideal for a range of reasons, including contributingto enhanced plant productivity for human consumption while at the sametime minimizing environmental damage. Minimizing environmental damageincludes obviating the need for artificial (synthetic) chemicalpesticides and artificial chemical fertilizers many of which are knownto persistent in the environment and whose presence is harmful toecosystems including humans, animals, insects and natural microbialpopulations.

The inventors contemplate that microbial compounds of the presentinventions can be combined or mixed, or into which they can be dissolvedor suspended or mixed. Suitable carriers which are well known includebut are not limited to water, solvents, aqueous solutions, such asmineral solutions, humic acid solution, etc., slurries, or dry powders;additional carriers include petrolatum products and diatomaceous earth(see e.g. U.S. Pat. No. 5,326,560; herein incorporated by reference).Other additional components, which may facilitate application of thecomposition to plants or seeds and which are well known, include but arenot limited to buffering agents, wetting agents, coating agents,abrading agents and other adjuvants, including but not limited topetroleum based materials or vegetable based materials, corn-starchencapsulated herbicide granules, citric acid, and complexpolysaccharides (see e.g., U.S. Pat. No. 5,945,377; herein incorporatedby reference) and alkali metal silicates (see e.g. U.S. Pat. No.5,183,477; herein incorporated by reference). In other preferredembodiments, the compositions further comprise components which aidplant growth and protection; such components include but are not limitedto fertilizer, insecticide, fungicide, nematocide, herbicide, and thelike. In yet other preferred embodiments, compositions further comprisecomponents that facilitate application of the composition to the plant,the plant part or the plant seed; such components include but are notlimited to buffering agents, wetting agents, coating agents, andabrading agents.

II. Amendments for Use in Formulations of the Present Inventions.

In some embodiments, liquid carriers of the present inventions furthercomprise a botanical compound. Indeed, a variety of botanical compoundsare contemplated, including but not limited to citrus pulp, preformedoil in water emulsion, corn cobs, corn meal, cracked corn, corn oil,edible oil, wheat bran, grape pomace, crude sorbitol, apple pomace, ricehulls, emulsified cottonseed oil, et cetera.

In some embodiments, liquid carriers comprise a spreader or wettingagent to ensure “wetting” of the surface to be sprayed. Examples ofwetting agents and spreaders include but are not limited to dried milk,powdered casein, gelatin, detergents, saponins, soaps, emulsifiers, suchas alkyl fenols, Tween 20, Tween 80, Sandovit, 9 D 207, Novémol,Pinolene 1882, Petro AG, Span 80, Triton X45, Triton N60, Triton X100,Triton X114, Triton GR7M, Triton 155, Atlox 848, Atlox 849, Tween 80,Atlox 3404/849, Atplus 448, Atplus 300 F and the like. The concentrationof wetting agent generally varies usually from 0.5% to 3.0% depending onthe concentration of the morphology and the surface properties of theactive ingredient.

Plants bred or engineered for resistance to numerous diseases caused byharmful soil borne microbes have not yet been developed. Growerscurrently depend on pesticides to fight some soil borne diseases whilefor other soil borne diseases there are no effective treatments.

The use of many pesticides causes environmental damage while there isincreasing amounts of public pressure against using them, somepesticides are expensive, difficult to apply, or not completelyeffective against soil borne pathogens. Moreover, pesticides mayindiscriminately kill both harmful and helpful soil microbes or presenta health risk to humans and animals. Cultural control methods such ascrop rotation may affect soil borne diseases very little since thepathogens that cause them attack a wide range of crops and can live insoil for a long time.

Current population of 6.5 billion human on this planet and the projectedneed to raise world food production by 110% in the next 50 years to meetthe growing food needs of the fast rising population, a decrease inarable land, and biofuels replacing food crops in many countries pose agreater challenge to food production industry worldwide (Triplett et al2007). Indeed, a massive global effort is needed to increase world foodproduction to keep pace with the needs of the rising human population.Using conventional approaches, high levels of nitrogenous fertilizersare needed to increase world food production to higher levels. Moreovermanufacture of nitrogen fertilizer requires fossil fuels as energysource resulting in the release of carbon dioxide, a greenhouse gas, andthus contributes to global warming. Furthermore, skyrocketing fossilfuel costs have pushed the cost of nitrogen fertilizers to recordlevels. This is quite significant considering the fact that nitrogenfertilizer is the highest farm cost for many food crops. Moreover thehigh use of the nitrogen fertilizer has adverse environmentalconsequences because of the leaching in to the ground water of nitrogencompounds such as nitrate, which at high levels poses a health hazard tohumans.

Furthermore, when soils become anaerobic, nitrate (NO₃) is reduced tonitrous oxide (N₂O) which is 319 times more potent than carbon dioxidein contributing to global warming. Hence, there is a vital need for aninnovative microbial product that greatly reduces or eliminated the needfor adding nitrogen fertilizer to a variety of food crops. This wouldcontribute not only to a substantial reduction in the costs of foodproduction but also would reduce the potential health and environmentalhazards that could result in the heavy use of nitrogen fertilizers.Moreover, a polymicrobial inoculate that would reduce or eliminate theneed for added chemical pesticides, by the including a Biocontrolagent(s), would not only give a cost advantage to the food producer butwould also eliminate another potentially important health andenvironmental hazards. Hence, microbial growth formulations that containdivers species of symbiotic nitrogen fixing bacteria to provide fixednitrogen needed by leguminous crops (such as soybeans, beans, peas,alfalfa, etc.) and free living nitrogen bacterial for providing fixednitrogen to nonleguminous crops (such as corn, rice, wheat, etc.) wouldbe highly desirable. Furthermore, the presence of microbes that serve asnatural bio-control agents against plant pathogens, other microbes thatstimulate plant growth by mobilizing minerals such as phosphorus, andthose that produce plant growth factors would be very desirable.

Furthermore, when soils become anaerobic, nitrate (NO₃) is reduced tonitrous oxide (N₂O) which is 319 times more potent than CO₂ incontributing to global warming. Hence, there is a vital need for aninnovative microbial product that greatly reduces or eliminated the needfor adding nitrogen fertilizer to a variety of food crops. This wouldcontribute not only to a substantial reduction in the costs of foodproduction but also would reduce the potential health and environmentalhazards that could result from the heavy use of nitrogen fertilizers.Moreover, a polymicrobial inoculate that would reduce or eliminate theneed for added chemical pesticides, by the including a Biocontrolagent(s), would not only give a cost advantage to the food producer butwould also eliminate another potentially important health andenvironmental hazard. Hence, microbial growth formulations that containdivers species of symbiotic nitrogen fixing bacteria to provide fixednitrogen needed by leguminous crops (such as soybeans, beans, peas,alfalfa, etc.) and free living nitrogen bacterial for providing fixednitrogen to nonleguminous crops (such as corn, rice, wheat, etc.) wouldbe highly desirable. Furthermore, the presence of microbes that serve asnatural bio-control agents against plant pathogens, other microbes thatstimulate plant growth by mobilizing mineral such as phosphorus, andthose that produce plant growth factors would be very desirable.

A successful polymicrobial inoculant should be efficacious andinexpensive (less than $1.00 per acre). Successful implementation of theinventions described herein are contemplated to reduce costs fornitrogen fertilizer usage by close to $30.00 per acre, much lower costsof food to the consumer, and less ground water and atmosphericpollution.

To the best of the inventors' knowledge, there is no polymicrobialinoculant in the marker that meets the comprehensive set of desiredcriteria described herein.

The inventors contemplate formulations of the present inventions aseco-friendly formulations, such that the production and use of theformulations of the present inventions are made with the health of theecology and the environment in mind. For example, the inventorscontemplate the use of the formulations of the present inventions forreducing the ecological impact of treating plants with harmfulchemicals, such as growth altering formulations, for example, growthmodulators, herbicides, and pesticides. Further, the inventorscontemplate the production and use of the formulations of the presentinventions as environmentally safe. Even further, the inventorscontemplate the production and use of the formulations of the presentinventions as biologically safe. In situations where the addition of apesticide a formulation of the present inventions would contribute toenhancement of plant productivity, a preferred embodiment aneco-friendly formulation of the present inventions further compriseorganic pesticides, such as a bio-herbicides, for example, caffeine,soybean oil, clove extracts; lemon juice, and vinegar acids, such as abiofungicide, for example, cinnamon. Thus, in preferred embodiments theformulations of the present inventions would be used in house plants,greenhouse plants, organic gardening and in fields for providing cropscertified as organic produce. Thus in further embodiments, theformulations of the present inventions would be permitted or certifiedfor use organic farming.

Preferred embodiments of eco-friendly formulations of the presentinventions do not preclude formulations comprising chemicals, such asherbicides and pesticides, including synthetic chemicals and artificialchemicals. Further, the addition of a chemical contributes to thesuccess of the plant enhancing characteristics of the presentinventions. Thus, in some embodiments, the amount of a chemical in aformulation at the point of application is lower than the amounttypically recommended for that application. In some embodiments, theinventors contemplate the addition of low quantities of chemicals toformulations of the present inventions, such that the quantities ofchemicals in solution when applied to soil and plants would be less thanthe amounts necessary to achieve equivalent plant growth enhancementswhen the chemical was used without the microbial mixtures of the presentinventions.

Compositions according to the embodiments are prepared by formulatingeach of the active ingredients, for example, a microbial isolate,separately and then mixing them together to prepare formulations of thepresent inventions. The point source (i.e. seed, plant, soil, and thelike) application amount varies with parameters such as weatherconditions, type of formulation, application timing, application method,application location, or the type of plant productivity desired, such asincrease in height, increase in leaf diameter, increase in yield, etcetera.

Various components of the microbial formulations are contemplated toinclude in addition to the microbial isolates, compounds and chemicals(also referred to as “amendments” or “soil amendments”) for allowingenhancement of plant productivity including but not limited to humates,surfactants, dispersants, chemical herbicides, nutrients, organic traceminerals, vitamins and natural polysaccharides and polypeptides, etc.These additional are typically in a suspension or solution whenformulated into the final composition, however, these components can beadded in dry form. Final formulations can be determined using routinegreenhouse and field-testing, for example, tests described herein.

The compositions of the present invention can be applied in any way toenhance plant growth and plant productivity. Compositions can be appliedundiluted or diluted, directly to the foliage of a plant, to seeds, suchas in seed dipping, seedling root dip or soaking the soil, such as asoil drench, or to other medium in which plants are growing or are to beplanted. The microbial formulations can be sprayed on, dusted on,applied in irrigation water, applied directly to the soil at the base ofa plant, applied directly to a plant, applied to a seed of a plant,applied to a root of a plant, and the like.

As a seed dip the microbial formulations are applied directly to theseed by several methods, such as by dipping seeds into a formulation,soaking the seeds in a formulation, soaking the soil prior to seedingwith formulation, and soaking the seeds in the soil with formulationwhere seeds were planted. In one embodiment, seeds, such as those oflegumes, such as peas and beans, and non-legumes, such as rice, corn,and sorghum, can also be soaked in a formulation from 30 minutes to 1hour prior to sowing.

As a foliar spray, the microbial formulations are applied to plantfoliage by methods commonly employed, such as conventionalhigh-gallonage hydraulic sprays, low-gallonage sprays, air-blast, aerialsprays and dusts. Application is contemplated be directed towards anypart of the plant including the foliage, base of the stems, branches,roots, or soil surrounding the roots. The microbial formulations of theinvention may also be injected into plants or sprayed onto vegetationusing electrodynamic spraying techniques or other low volume methods, orapplied by land or aerial irrigation systems. The dilution and rate ofapplication will be adjusted depending upon the type of equipmentemployed, the method and frequency of application desired, the crop, theclimate, and the weeds to be controlled. The amount of bacteria,nutrient matrix and additives can be adjusted to accommodate thegrowers' particular needs.

In some embodiments, the inventors contemplate protective and timereleased coatings where the microorganisms may be separatelyencapsulated in coatings, such as water soluble coatings and UV(ultraviolet) light protective coatings, e.g., dyed or undyed gelatinspheres or capsules, or by micro-encapsulation, such as by forming afree flowing powder encasing microorganisms. Examples of such coatingsare one or more of the following: gelatin, polyvinyl alcohol,ethylcellulose, cellulose acetate phthalate, or styrene maleicanhydride. The compositions can also be formulated in paraffin. Theseparately encapsulated microorganisms may be mixed directly with acarrier solution. In another case, encapsulated microorganisms may bemixed with non-encapsulated components. In one embodiment, encapsulationof the microorganisms includes nutrients as well as the microorganisms.

Aqueous suspension concentrates of largely insoluble solids may beprepared by ball or bead milling with a dispersing agent with asuspending agent included to stop the solid settling. Compositions to beused as sprays may be in the form of aerosols wherein the formulation isheld in a container under pressure of a propellant, e.g.fluorotrichloromethane or dichlorodifluoromethane. Water dispersiblepowders, emulsifiable concentrates and suspension concentrates willnormally contain surfactants, e.g. a wetting agent, dispersing agent,emulsifying agent or suspending agent. These agents can be cationic,anionic or non-ionic agents.

It is usually desirable, particularly in the case of foliar sprayformulations, to include adjuvants, such as wetting agents, spreadingagents, dispersing agents, stickers, adhesives and the like inaccordance with agricultural practices. Such adjuvants commonly used inthe art can be found in McCutcheon's “Emulsifiers and Detergents”,McCutcheon's “Emulsifiers and Detergents/Functional Materials” andMcCutcheon's “Functional Materials” all published annually by McCutcheonDivision of MC Publishing Company (New Jersey), herein incorporated byreference. The microbial formulations of the present invention typicallyhave one or more surfactants. The surfactants customarily employed inthe art of formulation of mixtures for foliar sprays or soil drenchesare described e.g. in “1985 International McCutcheon's Emulsifiers andDetergents” Glen Rock, N.Y. 07452, USA; “Encyclopedia of Surface ActiveAgents”, Chemical Publishing Co., Inc. New York, 1980; hereinincorporated by reference. Suitable surface-active compounds arenonionic, amphoteric and/or anionic surfactants having good emulsifying,dispersing and wetting properties. The term “surfactants” will also beunderstood as comprising mixtures of surfactants. Surfactants includeoil based spray additives, for example, certain mineral oil and naturalplant oil (such as soybean and rape seed oil) additives, or blends ofthem with other adjuvants.

For the preparation of emulsifiable concentrates, the compositions usedin the invention can be dissolved in suitable solvents or a mixture ofsolvents, together with an emulsifying agent that permits dispersion ofthe active compounds in water. Wettable powders suitable for spraying,can be prepared by admixing the composition with a finely divided solid,such as clays, inorganic silicates and carbonates, silicas, andincorporating wetting agents, sticking agents, and/or dispersing agentsin such mixtures.

When the microorganisms are dried or in spore form, they can beformulated into soluble powders or granules, which may containsurface-active agents to improve water dilution and preventcrystallization in a spray tank. The present compositions may beformulated to include a solid carrier to make, for example, tablets,dusts, and the like. Dusts are prepared by mixing a formulation of thepresent invention, or complexes thereof, with finely divided inertsolids, which can be organic or inorganic in nature. Inert materialsuseful for this purpose include botanical flours, silicas, silicates,carbonates and clays. When formulated into dustable powders or granules,fillers can be used such as kaolin, bentonite, kieselguhr, dolomite,calcium carbonate, talc, powdered magnesia, fuller's earth, gypsum,diatomaceous earth and china clay. Such granules can be preformedgranules suitable for application to the soil without further treatment.These granules can be made either by impregnating pellets of filler withthe active ingredient or by pelleting a mixture of the active ingredientand powdered filler. They may also be formulated in biodegradablepolymeric formulations to obtain a slow, controlled release of theactive substance. Concentrates should preferably be able to withstandstorage for prolonged periods and after such storage be capable ofdilution with water in order to form aqueous preparations which remainhomogeneous for a sufficient time to enable them to be applied byconventional spray equipment.

Although not required, this composition may contain additional additivesincluding fertilizer, insecticide, fungicide, nematacide, and mixturesthereof. Suitable fertilizers include (NH₄)₂NO₃. One example of asuitable insecticide is an organophosphate parasympathomimetic(Malathion) however any insecticide may be used in or with theformulations of the present inventions. One useful fungicide includesethanethiol (ethyl mercaptan; Captan) however any fungicide may be usedin or with the formulations of the present inventions.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate the process of the present invention. In addition, thebiocontrol agent can be applied to plant seeds with other conventionalseed formulation and treatment materials, including clays andpolysaccharides.

III. Induction of Systemic Resistance in the Host Plant and RootEnvironments.

A. Biocontrol Using Bacteria Species.

Numerous bacteria species are known for their capability to associatewith induction of or enhancement of systemic resistance to pathogens.However, not every bacteria isolate within such species has anequivalent capacity. For example, Paenibacillus species, are known fortheir ability to fix atmospheric nitrogen. Further, certainPaenibacillus isolates were shown to inhibit microbial pathogens in therhizosphere by producing antimicrobial substances. Thus they act asplant growth promoters as well as biocontrol agents (see, von der Weid,et al., 2003. Antimicrobial activity of Paenibacillus peoriae strainNRRL BD-62 against a broad spectrum of phyto pathogenic bacteria andfungi. J. Appl. Microbiol. 95:1143-1151; herein incorporated byreference). Further, in some embodiments, the inventors contemplateadding at least one or more known microbial isolate to formulations ofthe present inventions. Such isolates include but are not limited to,Paenibacillus brasiliensis 172, Paenibacillus peoriae deposited as NRRLBD-62, Paenibacillus polymyxa deposited as NRRL B37-A.

Further, the inventors contemplate isolating at least one novelPaenibacillus stain for providing antimicrobial plant enhancingcapabilities to formulations of the present inventions, such as thestrain described herein.

B. Biocontrol Using Fungal Species.

Fungal isolates demonstrate a range of activity such that while oneisolate causes disease in a plant, another isolate will protect a plantfrom disease. Specifically, Trichoderma isolates are used for biocontrolof plant diseases (Howell, 2003, Mechanisms employed by Trichodermaspecies in the biological control of plant diseases: The history andevolution of current concepts. Plant. Dis. 87:4-10; Benítez, et al.,2004, Biocontrol mechanisms of Trichoderma strains. Int Microbiol.December; 7(4):249-60. Review, all of which are herein incorporated byreference). General review on Trichoderma being plant symbionts: Harman,et al., 2004, Trichoderma species Opportunistic, avirulent plantsymbionts. Nature Rev. 44:43-56, herein incorporated by reference.Further, in addition to fungal species directly controlling other fungalspecies, certain fungal species were shown to induce increased pathogenresistance in plants (Shores, et al., 2005. Involvement of JasmonicAcid/Ethylene Signaling Pathway in the Systemic Resistance Induced inCucumber by Trichoderma asperellum T203, Phytopathology 95:76-84, hereinincorporated by reference).

A variety of pesticides belonging to different chemical classes are usedfor controlling disease affecting a variety of crops. Pesticides includebactericides and fungicides and a number of them are toxic to humans,some even at parts per billion. Furthermore, some of the pesticides arerecalcitrant to degradation and persist in the environment and enter thehumans/animal food chain. Such safety and environmental concerns aredriving the search for more environmentally friendly methods to controlplant disease that will contribute to the goal of sustainability inagriculture. Hence, bio-control agents (bio-pesticides) affectingdifferent plant disease are preferred. Many fungi and bacteria have beenexploited as biological control agents for inhibiting pathogenic fungi,bacteria and even nematodes and small insects. These soil-borne,non-pathogenic bacteria with the ability to antagonize fungalphytopathogens and thus prevent plant disease represents a realisticalternative to chemical fungicides. The bacteria that serve a biologicalcontrol agents (BCAs) and plant growth promoting rhizobacteria (PGPR) aswell as pseudomonads (which are known bio-pesticides are catabolicallyversatile, have excellent root-colonizing abilities, and have thecapacity to provide a wide range of metabolites that act against plantpathogens. Some soil microbes have been shown to elicit a diseaseresistant response in crop species, a phenomenon known as inducedsystemic resistant (ISR). Pseudomonas strains with the ability toproduce the antifungal metabolite 2,4-diacetylphloroglucinol (Phl) canbe isolated at high frequencies form soils suppressive to black root rotof tobacco and take-all disease of wheat. Given the ecological importantof Phl production, many of Pseudomonas BCAs, bio-control efficacy hasbeen liked to the production of this metabolite. In addition to Phlproduction, other secondary metabolites including pyoleueorin,pyrrolnitrin, and phenazines have been associated to bio-control (seereview Walsh et al., 2001). Bacillus spp., such as Bacillus subtilis areknown to suppress soil-borne fungal diseases and nematodes, producemetabolites that stimulate plant and root growth, and by colonizing theroot zone which excludes some pathogens by competitive exclusion.

IV. Induction of Modulation by Fungal Species for Enhancing NitrogenFixation in Leguminous Plants.

Nitrogen fixation in legumes is sensitive to soil acidity that can limitgrowth and persistence of rhizobia, the nitrogen fixing bacteria insoils (Graham, 1998, Biological dinitrogen fixation: Symbiotic. p.322-345. In Sylvia et al. (ed.) Principles and applications of soilmicrobiology. Prentice-Hall, Upper Saddle River, N.J.; hereinincorporated by reference). The range in sensitivity can be exemplifiedwhere fast-growing rhizobia that inoculate peas and lentils aregenerally more sensitive to soil acidity than strains that nodulatesoybean, although acid-tolerant rhizobia strains exist. Further, failureto nodulate roots in acid soils is due to both the lower numbers ofrhizobia and to the failure of attachment of rhizobia to the root hairs.Although it is common to lime acid soils, the areas involved especiallyin the Palouse and the cost and availability of limestone often limitthis practice. Alternative practices to combat soil acidity includeusing acid-tolerant inoculant rhizobia strains on host plants and limepelleting (coating) of inoculated seed. Liming also resulted inincreased nodulation in common bean and alfalfa (Buerkert et al, 1990;Pijnenberg and Lie, 1990, all of which are herein incorporated byreference).

Rhizobia are a group of symbiotic nitrogen fixing bacteria thatcontribute fixed nitrogen and promote production of leguminous plantsand have been widely used as inoculants. It was estimated that nitrogenfixing microbes associated with legumes grown on 25 million ha of landin Australia fix USD 3-4 billion worth of nitrogen annually (Brokwell,2004, Bullard et al. 2005). It is estimated that biological nitrogenfixation on a global scale reaches a value of 175 metric tons ofnitrogen fixed per year (Hubbel and Kidder, 2003). Of this total,symbiotic nitrogen fixation accounts for 20%. Grain legumes such ascowpea, peanut and soybean fix 250 of nitrogen per acre per year. Foragelegumes have been estimated to fix up to 250-300 lb nitrogen per acre.These statistics point to the importance of symbiotic nitrogen fixingorganism as a source of fixed nitrogen for the leguminous plants. Thebest known and most exploited symbiotic nitrogen fixing Rhizobia fallwith the genera: Rhizobium, Bradyrhizobium, Sinorhizobium (renamedEnsifer), Mesorhizobium, Allorhizobium, and Azorhizobium (O'Hara et al2003, Sorent 2001). It is of considerable inters that severalnon-Rhizobial species belonging to the alpha subgroup of Proteobacteriasuch as Methylobacerium, Phyulobacterium, Garobacerium, Hervaspirillumand Burkholderia have been reported form nodules and fix nitrogen inleguminous plants (Balachandar et al., 2007). Similar root noduleassociated N2 fixation has also been reported form somegamma-Proteobacteria (Benhizi et al, 2004).

In addition to symbiotic N2 fixing bacteria, there are a number offree-living N2 fixing bacteria. The Azospirillum group of organisms aremicroaerophilic nitrogen fixers commonly found in association with theroots of cereals such as rice, wheat and corn and certain forage grasses(Bashan et al., 2004). Azospirillum group makes significant contributionto nitrogen fixation and substantially decreases the level of nitrogenfertilizer needed for cereal crop production. Several studies indicatethat Azospirillum can increase the growth of various crops likesunflower, vegetables, cotton, wheat and rice (Bashan et al., 1989).Azospirillum canadense, A. lipoferum, A. oryzae, A. brasiliense are someof the known species that contribute to enhanced plant growth, nitrogenfixation and nutrient assimilation (Bashan et al., 2004). Other freeliving microbes that contribute to N2 fixation included: Acetobacter andHerbaspirillum associated with sugarcane, sorghum, and maize(Balachandar et al 2007, Boddey et al., 2000) and Alcaligenes, Bacillus,Enterobacter, Klebsiella and Pseudomonas strains associated with a rangeof crops such as rice and maize (Somasegaran and Hoben, 1994).Azotobacter, Beijerinkia and Clostridia are also recognized as freeliving N2 fixers (Polianskais et al., 2003).

Moreover, a number of plant growth promoting rhizobacteria are nitrogenfixers and five a positive growth response that has been attributed tosecondary growth promoting compounds, such as plant growth hormone,produced by these organisms (Polianskais et al., 2003). Paenibacillus,Burkholderia, and alpha, beta, and gamma) Proteobacteria have also beenreported to fix nitrogen or otherwise stimulate plant growth.Paenibacillus polymyxa increase both numbers and nodulation by Rhizobiumspecies (Petersen et al., 1996). Phosphate-solubilizing bacteria (PSB)are also important as they have been reported to produce organic acidsand convert insoluble P compound to soluble for uptake by the plant.Important Phosphate solubilizing organisms include but are not limitedto Pseudomonas, Bacillus, Azospirillum, Rhizobium, Alkaligenes,Paenibacillus, and Penicilluim digitatum (Rodriguez and Fraga, 1999).PSB are of great value in allowing the use of less expensive P sources(Sundara et al., 2002).

Many of the plant growth promoting bacteria such as Bacillus spp. andPseudomonas spp. have also been reported to posses multi beneficialcharacteristics such as nutrient recycling, nutrient uptake andphytohomone production (Rai., 2006).

So although other Tichoderma spp. were known for treating plants toprotect certain plants against certain pathogens, the inventorsdemonstrate herein that solutions consisting of multiple Tichoderma spp.of the present inventions induce nodulation of pea plant roots. Thusadditional embodiments encompass the use of formulations comprisingTrichoderma species of the present inventions for enhancing plantproductivity, in particular legume formation and increased nitrogenfixation. In some embodiments, the formulations consist of Trichodermaspecies of the present inventions for enhancing plant productivity.

V. Formulations.

The inventors contemplate a variety of formulations for use in thepresent inventions. In one embodiment, the inventors contemplate amicrobial mixture for use in a variety of soil environments. Such amixture would comprise isolates capable of surviving under a variety ofenvironments for conferring a benefit to a plant and to populations ofplants.

A. Providing Formulations of the Present Inventions from DepositedMixtures.

The inventors contemplate the use of deposited mixtures, for example,F2A and F2B, for providing formulations of the present inventions. Thefollowing is a contemplated procedure for thawing, growing and mixingthe microorganisms for use in the present inventions, for example,Sumagro-4, 5 and 2.

Methods of thawing and growing lyophilized bacteria and fungi arecommonly known, for example, Gherna, R. L. and C. A. Reddy. 2007.Culture Preservation, p 1019-1033. In C. A. Reddy, T. J. Beveridge, J.A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. Snyder, eds.American Society for Microbiology, Washington, D.C., 1033 pages; hereinincorporated by reference. Thus freeze dried liquid formulations andcultures stored long term at −70° C. in solutions containing glycerolare contemplated for use in providing formulations of the presentinventions.

Sumagro-4 (F4) fungal formulations are made directly from fungalmixtures of F4 as described in the Examples. Formulations of thebacteria isolate mixtures shown for Sumagro-2 are also made directlyfrom stored bacteria mixtures containing the bacteria listed forSumagro-2 as described in the Examples. Sumagro-2 is provided bycombining the mixture of bacteria isolates and mixture of fungalisolates according to descriptions provided in the Examples herein.

B. Re-Isolating Microbes of the Present Inventions for ProvidingIsolates for Use in Additional Formulations, as Individual Isolates,Mixtures and Formulations.

Alternatively, formulations of the present inventions and additionalformulations, including formulations of individual isolates andformulations of a variety of combinations of the microbes arecontemplated. These formulations may be provided by re-isolating eachmicrobe from the formulations described herein. Even further, theseformulations may be provided by re-isolating individual isolates frommixtures, such as F2A and F2B. Specifically, bacteria may re-isolatedusing the isolation methods and identification methods as describedherein and in: C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A.Marzluf, T. M. Schmidt, and L. R. Snyder (eds.). Methods for General andMolecular Microbiology, Am. Soc. Microbiol., Washington, District ofColumbia. In particular, individual bacteria isolates may be provided byserial diluting mixtures and plating for isolation of single coloniesfollowed by growing isolates for 16s rDNA identification. Further,individual bacteria isolates may be provided from formulations, such asF2, and soil samples treated with formulations of the presentinventions, such as F1, F2, etc. Individual isolates matching thoseprovided herein, may be identified with 16s RNA PCR techniques asdescribed in the Examples. Fungus isolates may also be re-isolated frommixtures, formulations and treated soil. These isolates are seriallydiluted, plated, grown and identified as described in the Examples.

These re-isolated bacteria and fungal isolates are contemplated for usein a variety of formulations. Specifically, individual isolates arecontemplated for further testing and for use individually and incombinations. In one embodiment, the microbial isolate is combined withother isolates provided herein for providing formulations of the presentinventions. In another embodiment, the microbial isolate is combined insubformulations of the present inventions. In even further embodiments,the microbial isolate is combined in any combination with other isolatesin order to provide formulations of the present inventions.

VI. Applications.

In addition to field and greenhouse applications, the inventors furthercontemplate using formulations of the present inventions for enhancingplant growth in house plants, on lawns, sports fields, highway areas,such as medians, roadsides, exit and entrance lanes, and to encouragegrowth of wildlife populations, such as National Forests, wildlifeprotection areas, private reserves, and the like. Further, the inventorscontemplate using formulations of the present inventions for replantingareas with severely altered or damaged soils, including soils with highacidity.

In some embodiments, the inventors contemplate adding formulations ofthe present inventions in mulching material for treating plants, for oneexample, mulching material for blueberry plants.

In some embodiments, the inventors contemplated using formulations ofthe present inventions for increasing biomass of plants for use inalternative energy programs such as biofuel production. The inventorscontemplate the use of their formulations on large tracts of switchgrass (prairie grass) for increasing biomass. In particular sinceformulations of the present inventions are contemplated to boost biomassproduction from single and multiple stands of prairie grass, theseformulations may overcome the limitations reported as “Pure switch grassstands may not be best for ethanol By DALE HILDEBRANT, Farm & RanchGuide Friday, Apr. 13, 2007 9:18 AM: CDT. “According to Hill, theirstudy found that mixtures of 16 native prairie species produced 238percent more energy on average than a single prairie species such asswitch grass and as an added bonus, the stands made up of the plantmixtures removed large amounts of carbon dioxide from the air and storedit in the soil, but that the single species stands did not.”

In particular, the inventors contemplate the use of formulations of thepresent inventions for organic farming and production of crops fordesignation as organic products. Even further, the inventors contemplatethe use of formulations of the present inventions for treating plants inareas of low water or drought, such that formulations of the presentinventions would enhance drought resistance in treated plants.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmicrobiology, botany, biochemistry, chemistry, molecular biology, plantbiology, plant disease, and plant pathogens, or related fields areintended to be within the scope of the following claims.

EXPERIMENTAL

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof.

In the experimental disclosures which follow, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomole); pmol (picomole); g (gram); mg (milligram); μg (microgram);ng (nanograms); pg (picogram); L and l (liter); ml (milliliter); μl(microliter); cm (centimeter); mm (millimeter); μm (micrometer); nm(nanometer); U (unit); min (minute); s and sec (second); k (kilometer);deg (degree); and ° C. (degrees Centigrade/Celsius), potato dextroseagar (PDA) and tryptone-mannitol-yeast extract (TMY).

As used herein, an exemplary comparison commercial product was NutraGro(Nutragro), designated NG, (BioSoil Enhancers, Inc., originally,Bio-Solutions Manufacturing, Inc.) which was a solution providing “mixedcultures of beneficial microbes, macro and micronutrients, and bioactivesubstances to promote soil health and crop potential.” (Market Wire,April, 2006, at//findarticles.com/p/articles/mi_pwwi/is_(—)200604/ai_n16121689, hereinincorporated by reference).

As used herein, one of the exemplary carrier solutions was HumaGro(Humagro), designated HG, (BioSoil Enhancers, Inc., originally,Bio-Solutions Manufacturing, Inc.) a commercially available productwhich contained “nutrients, organic matter and humic acid, which helpssoil, microbes, and plants.” (Market Wire, April, 2006, at//findarticles.com/p/articles/mi_pwwi/is_(—)200604/ai_n16121689, hereinincorporated by reference). As used herein, another exemplary carriersolution was a mineral solution as described herein.

EXAMPLE I

The inventors' objective was to develop an efficacious, eco-friendly,and cost-effective formulation suitable for enhancing the productivityof a broad spectrum of crops such as cereals, pulses, vegetable,horticultural and floricultural corps. To this end, the inventors wereable to isolate multifunctional microbes, useful for constructing asuitable microbial inoculant formulation(s), form root nodules ofvarious legume, rhizosphere soils and soils collected from temperate,tropical and sub-tropical regions. The results indicate a wide range ofphenotypic diversity of the isolates. The inventors then constructed atleast two microbial inoculant formulation designated F1 and F2, (seeExamples below) each with a distinct set of microbes and tested theirefficacy on various crops under greenhouse condition and fieldconditions. Both conditions confirmed enhanced growth and yield of abroad spectrum of legumes, cereals, and vegetable crops as described inthe Examples.

Materials and Methods.

A. Providing Soil Microbial Isolates.

The inventors collected soil samples from a variety of global locationsincluding the United States of America. These samples included soilremoved from the rhizosphere and root nodules of a variety of plants.When the inventors applied commonly known microbial isolation proceduresto these soils, the inventors obtained a variety of microbes includingbacteria and fungi that were further subjected to conventional isolationtechniques for providing pure isolates as described herein.

1. Isolation of Rhizobacteria from Soil.

In general, a randomly collected 30 g soil sample was obtained at eachselected site then air-dried, made free of any pellets, and sieved toremove pebbles and clumped material. The resulting fine powdery soil wassuspended in sterile double distilled water and subjected to serialdilutions (10⁻¹ through 10⁻⁷). One ml soil suspension from each of thedilutions from 10⁻⁴ to 10⁻⁷ was placed on nutrient agar plates andincubated at 28° C. (spread plate method). Specific growth media andincubation conditions were used to isolate a variety of groups ofbacteria from the soil. Dilutions and platings were carried out understerile conditions.

Standard microbial enrichment technique (C. A. Reddy, T. J. Beveridge,J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. Snyder (eds.).Methods for General and Molecular Microbiology, Am. Soc. Microbiol.,Washington, District of Columbia) was followed to isolate each type ofbacteria. For example, for Azospirillum sp. isolates, Nitrogen-freemalic acid semisolid medium was used for enrichment. Observations of thedevelopment of subsurface pellicles during incubation and change ofcolor of the medium from light yellow to blue was suggestive of thepresence of colonies of Azospirillum. For phosphobacteria isolates,plating of the soil sample was done on Sperbers agar. Those colonieswith halos around them were selected and screened for solubilization oftricalcium phosphate. Pikovskays's medium was another general purposemedium for selection of phosphate-solubilizing bacteria. For Bacillussp. isolates the Nutrient medium was used.

Bacteria associated with the plant rhizosphere are referred to asrhizobacteria. For rhizobacteria isolates, plant roots along withadherent soil were carefully removed from the soil, washed twice withsterile distilled water, centrifuged with the resulting pelletresuspended in buffer and was subject to serial dilution technique asdescribed above. Pseudomonas, Azospirillum and Bacillus sp. wereisolated from the rhizosphere samples. Selected agricultural soils werealso used for isolating phosphobacteria, Acetobacter and some Bacillusspecies.

2. Isolation of Fungal (Trichoderma) Species.

Several species of Trichoderma for use in the present inventions wereisolated from diverse soil samples collected from cultivated agriculturelands, tropical forest soils and sub tropical forest soils. Initialisolations were carried out using dilution plate technique on standardpotato dextrose agar medium. Isolated strains were purified by singlespore colony and the taxonomic classification was based on colony color,rate of growth, and macro and microscopic features of mycelia, shape andsize of conidia and phialids.

The isolated Trichoderma species were screened for their bio-controlpotential against known plant pathogenic fungi using standard dualculture plate technique. Trichoderma strains were also tested for theirsaprophytic competency in soil. Seven of the best isolates were chosenby the inventors for use in formulations of the present inventions.

For long time storage, a sterile soil sample was inoculated with anisolate and stored at 4° Celsius. The Trichoderma isolates used informulations of the present inventions were: Trichoderma harzianum (3strains), Trichoderma viride (2 strains), and a strain of Trichodermalongibrachiatum, and a strain of Trichoderma virens.

Fungal inoculum was prepared in Potato dextrose broth in three steps:first in small tubes, then scaled up to small Ehrlenmeyer flasks, andthen up to 2 liter Ehrlenmeyer flasks. A fungal mat was collected byfiltration using a sterile Buchner funnel with the fungal mat filteredout through cheese-cloth after which the conidia were collected throughcentrifugation at 5000×g for 10 minutes. Both mycelial mat and conidialpellet were thoroughly blended in a sterile blender before mixing withthe bacterial inoculants in preparing the final formulations.

B. Preparation of Microbial Formulations from Soil Isolates.

Pure stock cultures of bacteria and fungi were grown and then used inthe preparation of microbial formulations. Stock cultures of isolateswere stored as streaks on tryptone-mannitol-yeast extract (TMY) agarslants at 40° C. until further use.

A liquid carrier containing was used in preparing microbialformulations. In particular, the inventors used a 12% humic acid (v/v)solution at pH 7.0, such as a Humagro solution, in designatedformulations, while in other formulations the inventors used a mineralsolution.

1. Preparation of Sumagro 1 (F1) in Humagro.

The following are exemplary steps for preparing a Sumagro 1 formulationof the present inventions.

Thirty-three bacterial isolates and 7 fungal isolates (listed inTable 1) were used for preparing Sumagro 1. Culture inoculations andtransfers were done under standard aseptic conditions. Specifically, aloop of bacterial culture from a stock slant of the bacterial isolate inTable 1 was used to inoculate 5 ml of TMY liquid broth in 18×150 mmfoam-plugged culture tubes and incubated on a shaker at 30° C. for 16 hrfor Bacillus and 30 hr for Rhizobium cultures. One ml from the 5 mlculture was then used to inoculate 75 ml of TMY broth in a 150 mlEhrlenmeyer flask and incubated as described above. The 75 ml culturewas then used to inoculate one liter of TMY broth in 2-liter Ehrlenmeyerflasks and incubated as described above.

The 2-liter flask cultures were harvested by centrifugation at 7000 rpmfor 10 min at 4° C. using sterile 500 ml screw-capped centrifugebottles. The cell pellets from each centrifuge bottle was suspended in10 ml of the humic acid carrier solution (pH 7.0) mentioned above. Cellsuspensions of all the 33 bacterial cultures prepared in this mannerwere pooled together.

The seven Trichoderma strains were cultured individually in potatodextrose broth (rather than in TMY broth) and the same cultivation stepsdescribed above for bacterial cultures were used. Trichoderma fungal matwas collected by centrifugation as described above and homogenizedthoroughly in 50 ml of the carrier solution using a sterile stainlesssteel blender. The Trichoderma suspensions from each of the sevencultures were then pooled together.

The pooled bacterial suspension and the Trichoderma suspension was mixedtogether and made up to a total volume of 5 liters using the humic acidcarrier described herein. This formulation consisting of 33 bacterialstrains and seven Trichoderma strains was designated as Sumagro 1 (F1).

TABLE 1A Sumagro-1 (F1) Cultures Used in Growth-Enhancing MicrobialFormulations. Sumagro-1 (F1) Cultures Genus species designation  1.Bacillus sp. RG-S  2. Bacillus sp. BL  3. Bacillus sp. RG-2L  4.Bacillus sp. Ph.L-1  5. Bacillus sp. Ph.L  6. Ensifer meliloti FD  7.Rhizobium trifolii FD  8. Azorhizobium caulinodans KN  9. Rhizobium sp.RLG1 10. Azorhizobium sp. RLG2 11. Azorhizobium sp. RLG3 12. Rhizobiumsp. RLG4 13. Rhizobium sp. RLG5 14. Rhizobium sp. RLG6 15. Azorhizobiumsp. RLG7 16. Rhizobium sp. RLG8 17. Azorhizobium sp. RLG9 18. Rhizobiumsp. RLG10 19. Rhizobium sp. RLG11 20. Rhizobium sp. Ph 21. Rhizobium sp.B 22. Rhizobium sp. Ph.P 23. Rhizobium sp. L-26 24. Rhizobium sp. L-2725. Rhizobium sp. L-30 26. Rhizobium sp. L-32 27. Rhizobium sp. B5 28.Bacillus sp. B6 29. Rhizobium sp. B1A 30. Rhizobium sp. B2 A1 31.Rhizobium sp. M 32. Rhizobium sp. S12 33. Rhizobium sp. S13 34.Trichoderma virens 3107 35. Trichoderma viride LK 36. Trichoderma viride3116 37. Trichoderma harzianum 3147 38. Trichoderma harzianum G 39.Trichoderma harzianum LK 40. Trichoderma longibrachiatum 3108 40 Totalcultures Sumagro-1 Revised Genus species (F1) Genus species culturedesignation Cultures designation designation based upon 16s rDNA  1.Bacillus sp. RG-S na na  2. Bacillus sp. BL na na  3. Bacillus sp. RG-na na 2L  4. Bacillus sp. na na Ph.L-1  5. Bacillus sp. na na Ph.L  6.Ensifer meliloti RMEL1 Ensifer (Sinorhizobium) FD meliloti RM1  7.Rhizobium RTRF1 Rhizobium trifolii FD leguminosarum bv. trifolii RT1  8.Azorhizobium AZOR1 Azorhizobium caulinodans KN caulinodans AZ1  9.Rhizobium sp. RLNG1 Pseudomonas sp. RL1 RLG1 10. Azorhizobium RLNG2Pantoea (Enterobacter) sp. RLG2 agglomerans RL2 11. Azorhizobium RLNG3Stenotrophomonas sp. RLG3 maltophila RL3 12. Rhizobium sp. RLNG4Stenotrophomonas RLG4 maltophila RL4 13. Rhizobium sp. RLNG5Stenotrophomonas RLG5 maltophila RL5 14. Rhizobium sp. RLNG6 Bacillussubtilis RL6 RLG6 15. Azorhizobium RLNG7 Bacillus subtilis RL7 sp. RLG716. Rhizobium sp. RLNG8 Pseudomonas sp. RL8 RLG8 17. Azorhizobium RLNG9Bacillus subtilis RL9 sp. RLG9 18. Rhizobium sp. RLNG10 StenotrophomonasRLG10 maltophila RL10 19. Rhizobium sp. RLNG11 Pseudomonas sp. RL11RLG11 20. Rhizobium sp. Ph 21. Rhizobium sp. B na na 22. Rhizobium sp.na na Ph.P 23. Rhizobium sp. na na L-26 24. Rhizobium sp. na na L-27 25.Rhizobium sp. na na L-30 26. Rhizobium sp. na na L-32 27. Rhizobium sp.na na B5 28. Bacillus sp. B6 na na 29. Rhizobium sp. na na B1A 30.Rhizobium sp. na na B2 A1 31. Rhizobium sp. M na na 32. Rhizobium sp. nana S12 33. Rhizobium sp. na na S13 34. Trichoderma TV LK Trichodermaviride LK virens 3107 35. Trichoderma TV 3116 Trichoderma viride virideLK 3116 36. Trichoderma TVs 3107 Trichoderma virens viride 3116 3107 37.Trichoderma TH 3147 Trichoderma harzianum 3147 harzianum 3147 38.Trichoderma TH LK Trichoderma harzianum G harzianum LK 39. TrichodermaTH G Trichoderma harzianum LK harzianum G 40. Trichoderma TLB 3108Trichoderma longibrachiatum longibrachiatum 3108 3108 40 Total cultures

2. Preparation of Sumagro 2 (F-2) in Humagro.

A formulation consisting of 14 bacterial strains and 7 Trichodermastrains, where the Trichoderma strains were added at twice theconcentration in Sumagro 1, was prepared and designated Sumagro 2 (F2).See, Table 2.

Sumagro 2 was prepared as described for Sumagro 1, with the exception ofreducing the number of bacterial cultures to 14 (rather than 33 as usedin Sumagro 1). Further, the seven Trichoderma cultures were added attwice the concentration used in Sumagro 1 (see Table 2 listing Sumagro 2cultures). Briefly, bacterial and fungal cultures were grown,centrifuged, resuspended, and pooled together as described above forpreparing Sumagro 1. The pooled suspension was then made up to 5 litersusing the humic acid carrier and stored at 4° Celsius.

TABLE 2A Sumagro-2 (F2) Cultures Used in Growth-Enhancing MicrobialFormulations. Sumagro 2 (F2) Cultures Genus species designation 1.Ensifer meliloti FD 2. Rhizobium trifolii FD 3. Azorhizobium caulinodansKN 4. Rhizobium sp. RLG1 5. Azorhizobium sp. RLG2 6. Azorhizobium sp.RLG3 7. Rhizobium sp. RLG4 8. Rhizobium sp. RLG5 9. Rhizobium sp. RLG610. Azorhizobium sp. RLG7 11. Rhizobium sp. RLG8 12. Azorhizobium sp.RLG9 13. Rhizobium sp. RLG10 14. Rhizobium sp. RLG11 15. Trichodermavirens 3107 16. Trichoderma viride LK 17. Trichoderma viride 3116 18.Trichoderma harzianum 3147 19. Trichoderma harzianum G 20. Trichodermaharzianum LK 21. Trichoderma longibrachiatum 3108 21 Total CulturesNote: Concentration of each of the Trichoderma strains were doubled inSumagro 2 based on the rationale that Trichoderma strains are potentinhibitors of fungal pathogens, induce host resistance to bacterial andfungal plant pathogens, induce production of growth-stimulating hormonalsubstances such as auxins and cytokinins, and the inventors observed inpreliminary experiments that they stimulate nodulation by soildiazotrophs (Benitez, et al. 2004. Biocontrol mechanisms of Trichodermastrains. International Microbiology 7: 249-260; herein incorporated byreference). Theses product formulations appear stable over a 6-monthperiod at ambient temperature.16S rDNA Sequencing for Molecular Identification of Bacterial Isolatesof the Present Inventions.

This Example describes an exemplary method for determining the Genusspecies of the bacterial isolates of the present inventions.

Polymerase chain reaction (PCR) amplification of DNA from each givenisolate for 16S rDNA sequencing was done using established procedures(Mignard and Flandrois, 2006; Pandey et al., 2004; Petti, 2007; each ofwhich are herein incorporated by reference in their entirety). The 16SrRNA gene from the total DNA of a bacterial strain was amplified withbacterial universal forward primer 8F (number of bases 20) with thesequence: AGAGTTTGATCCTGGCTCAG (SEQ ID NO: 1). The reverse primer (1492Rwith 19 bases) had the sequence: GGTTACCTTGTTACGATT (SEQ ID NO: 2).These primers were obtained from Macromolecular Structure Facility,Michigan state University. Polymerase chain reaction amplificationcycles commenced with an initial denaturation at 95° C. for 3 minfollowed by 30 cycles of 30 sec each at 95° C., 30 sec at 55° C., and 45sec at 72° C., with a final extension of 10 min at 72° Celsius. PCRproducts were checked by electrophoresis on 0.8% agarose gel, purifiedusing QIA quick spin PCR purification kit (following the protocol inQuiagen quick spin handbook). Purified PCR products were checked byelectrophoresis on 0.8% agarose gel. The PCR purified products were thensequenced using 531R primer (number of bases 18) with the sequence:TACCGCGGCTGCTGGCAC (SEQ ID NO: 3). Sequence data were aligned andcompared with available standard sequences of bacterial lineage in theNational Center for Biotechnology Information Gene Bank(ncbi.nlm.nih.gov/blast/Blast.cgi) using BLAST search program (See, forreview, McGinnis S., & Madden T. L., (2004) Nucleic Acids Res.32:W20-W25; Ye, et al., (2008) Nucleic Acids Res. 34:W6-W9; Johnson etal., (2008) Nucleic Acids Res. 36:W5-W9, each of which are hereinincorporated by reference in their entirety.

The validity of the procedure was established by using DNA from knownRhizobium and Azorhizobium cultures. The procedure described was usedfor the identification of all the bacterial strains in F1 and F2formulations (Mignard and Flandrois. 2006. J. Microbiol. Methods67:574-581; Pandey, et al., 2004. Current Sci. 86: 202-207; Petti, 2007.Clin. Infect. Dis. 44:1108-1114, and Macrae, (2000) Brazilian Journal ofMicrobiology 31:77-82, each of which are herein incorporated byreference in their entirety).

TABLE 2B Sumagro-2 (F2) Genus identification of Cultures Used in Growth-Enhancing Microbial Formulations.* F2 Cultures: Sumagro Initial GenusRevised Revised Genus 2 (F2) species designation designation speciesdesignation** Cultures from Table 2A RLNG1 Pseudomonas sp. RL1 4.Rhizobium sp. RLG1 RLNG2 Pantoea 5. Azorhizobium sp. (Enterobacter) RLG2agglomerans RL2 RLNG3 Stenotrophomonas 6. Azorhizobium sp. maltophilaRL3 RLG3 RLNG4 Stenotrophomonas 7. Rhizobium sp. RLG4 maltophila RL4RLNG5 Stenotrophomonas 8. Rhizobium sp. RLG5 maltophila RL5 RLNG6Bacillus subtilis RL6 9. Rhizobium sp. RLG6 RLNG7 Bacillus subtilis RL710. Azorhizobium sp. RLG7 RLNG8 Pseudomonas sp. RL8 11. Rhizobium sp.RLG8 RLNG9 Bacillus subtilis RL9 12. Azorhizobium sp. RLG9 RLNG10Stenotrophomonas 13. Rhizobium sp. RLG10 maltophila RL10 RLNG11Pseudomonas sp. 14. Rhizobium sp. RLG11 RL11 RTRF1 Rhizobium 2.Rhizobium trifolii FD leguminosarum bv. trifolii RT1 RMEL1 Ensifer 1.Ensifer meliloti FD (Sinorhizobium meliloti) RM1 AZOR1 Azorhizobium 3.Azorhizobium caulinodans AZ1 caulinodans KN TV LK Trichoderma viride 16.Trichoderma viride LK LK TV 3116 Trichoderma viride 17. Trichodermaviride 3116 3116 TVs 3107 Trichoderma virens 15. Trichoderma virens 31073107 TH 3147 Trichoderma 18. Trichoderma harzianum 3147 harzianum 3147TH LK Trichoderma 20. Trichoderma harzianum LK harzianum LK TH GTrichoderma 19. Trichoderma harzianum G harzianum G TLB 3108 Trichoderma21. Trichoderma longibrachiatum 3108 longibrachiatum 3108 *This group oforganisms are the same as shown in Table 2A however for some isolatesthe Genus was re-assigned based upon DNA identification.**Identification of bacterial species was confirmed by 16S rDNAsequencing

TABLE 2C Sumagro-5 (F5; F2A) Genus identification of Cultures Used inGrowth-Enhancing Microbial Formulations.* F2A (F5) As Initial GenusCultures: Genus species listed species designation Abbreviationdesignation** for F2 from Table 2A RLNG1 Pseudomonas sp. 4. Rhizobiumsp. RLG1 RL1 RLNG2 Pantoea 5. Azorhizobium sp. RLG2 (Enterobacter)agglomerans RL2 RLNG3 Stenotrophomonas 6. Azorhizobium sp. RLG3maltophila RL3 RLNG4 Stenotrophomonas 7. Rhizobium sp. RLG4 maltophilaRL4 RLNG5 Stenotrophomonas 8. Rhizobium sp. RLG5 maltophila RL5 RLNG6Bacillus subtilis RL6 9. Rhizobium sp. RLG6 RLNG7 Bacillus subtilis RL710. Azorhizobium sp. RLG7 RLNG8 Pseudomonas sp. 11. Rhizobium sp. RLG8RL8 RLNG9 Bacillus subtilis RL9 12. Azorhizobium sp. RLG9 RLNG10Stenotrophomonas 13. Rhizobium sp. RLG10 maltophila RL10 RLNG11Pseudomonas sp. 14. Rhizobium sp. RLG11 RL11 RTRF1 Rhizobium 2.Rhizobium trifolii FD leguminosarum bv. trifolii RT1 RMEL1 Ensifer 1.Ensifer meliloti FD (Sinorhizobium meliloti) RM1 AZOR1 Azorhizobium 3.Azorhizobium caulinodans AZ1 caulinodans KN

Exemplary Formulations for Greenhouse Experiments.

The dose per pot in the Greenhouse was proportionally the same as thatused in the recommended dose for field applications.

In the Greenhouse, F1 and F2 containing 10¹⁴ microbes/ml were used. Onepart of F1 or F2 was added to 4 parts of a humate carrier, inparticular, Humagro. Pots sizes were 4×5″ with a 500 gram soil volume.

Exemplary Formulations for Field Trials.

Five liters of Sumagro microbial formulation containing a total of 10¹¹microbial cells [100 billion cells, approximately 2×10⁷ cells/ml] wereused per acre. One liter of Sumagro 1 or Sumagro 2 were mixed with fourliters of Humagro and applied per (one) acre.

Control solutions for field trials were the commercial formulation,Nutragro, comprising a microbial mixture. One liter of Nutragro wasmixed with 4 liters of Humagro and applied per one acre of soil.

A total of three applications (at least Two) are recommended for onecrop season. One at the time of sowing and second application one monthafter the first application. The foliar application was recommended as aspray at the base of the stem or nearer to the root zone.

3. Preparation of Sumagro-3 and Sumagro-4 in Humagro.

A formulation consisting of 10 bacterial strains and 4 Trichodermastrains was prepared as described for Sumagro 1, and designated Sumagro3 (F-3). See, Table 3 for microbial strains in Sumagro 3. An additionalformulation consisted of seven Trichoderma strains was prepared asdescribed for Sumagro 1 but without adding bacterial strains, see Table4 for Trichoderma strains in Sumagro 4 (F-4).

TABLE 3A Sumagro-3 Cultures Used in Growth-Enhancing MicrobialFormulations. Sumagro 3 Genus species 1. Bacillus sp. LK(phosphate-solubilizing bacterium) 2. Bacillus subtilis LK(phosphate-solubilizing bacterium) 3. Pseudomonas fluorescens -(stimulates growth by increasing nutrient availability) 4.Azospirillum - free living diazotroph 5. Acetobacter sp. LK- free livingdiazotroph 6. Rhizobium phaseoli - symbiotic diazotroph 7.Bradyrhizobium japonicum - symbiotic diazotroph 8. Rhizobium melilotiFD - symbiotic diazotroph 9. Rhizobium trifolii FD - symbioticdiazotroph 10. Azorhizobium caulinodans KN - symbiotic diazotroph 11.Trichoderma harzianum 3147 12. Trichoderma viride 3116 13. Trichodermavirens 3107 14. Trichoderma longibrachiatum 3108

TABLE 4 Sumagro-4 (F2B) Fungal Cultures Used as Mixtures and in Growth-Enhancing Microbial Formulations. Sumagro 4 Abbreviation Genus SpeciesName* 1. TH 3147 Trichoderma harzianum 3147 2. TH G Trichodermaharzianum G 3. TH LK Trichoderma harzianum LK 4. TV 3116 Trichodermaviride 3116 5 TV LK Trichoderma viride LK 6. TVs 3107 Trichoderma virens3107 7. TLB 3108 Trichoderma longibrachiatum 3108 *identified asdescribed herein.

Exemplary characterizations of microbial isolates for use in providingbasic nutrients in formulations of the present inventions.

Nitrogen Fixation Tests: The inventors evaluated bacterial isolates ofthe present inventions for their capability to fix free Nitrogen(atmospheric nitrogen). For this example, bacterial isolates were platedonto nitrogen-free glucose medium. Capability for growth on this mediumindicated that the organism was able to fix nitrogen. Exemplary resultsare shown in Table 5.

Phosphate Solubilization tests: Phosphate solublizing Bacteria (PSB)were identified by visual observation of bromophenol blue productionusing NBRI-BPB growth medium (for example, methods in Curr Microbiol.2001 July; 43(1):51-6, herein incorporated by reference).

TABLE 5 Identification of free Nitrogen fixing and PhosphateSoluabilizing Bacteria Isolates of the present inventions. Blue colorproduced on NBRI-BPB growth F2 Growth on medium for indicating CULTURESminus Nitrogen Phosphate (Strain) broth medium* Solubilization** RLNG1++/+ − RLNG2 −/− − RLNG3 −/− − RLNG4 −/− − RLNG5 +/+ + RLNG6 +/+ − RLNG7+/+ − RLNG8 +/+ − RLNG9 ++/+ + RLNG10 +/++ + RLNG11 +/+ − R. TRIFOL +/++− R. MELI ++/++ − AZORHIZO ++/++ − *−/− = no growth, +/+ indicates avery low level growth thus of nitrogen fixation, any combination of +/++and ++/+ indicates a moderate level of nitrogen fixation, while ++/++indicates a high level of nitrogen fixation. **− indicates no blueproduction while + indicates blue color production.

4. Preparation of Microbial Formulations in Mineral Solution Carriers.

a. Microbial Formulations with Mineral Solutions.

The inventors further tested their microbial formulations using anexemplary mineral based carrier solution.

Preparation of 1 Liter of a working mineral solution (MM) was made bythe addition of 50 ml of Macro-A, 50 ml of Macro-B stock solutions and 1ml of the trace element stock solution with the volume brought up to 1Liter. The following stock solutions used for these formulations wereprepared individually as a 10× concentration stock solution in doubledistilled water and sterilized by autoclaving. Macro-A: NH₄NO₃ 1.0 g/L,KH₂PO₄ 0.3 g/L, K₂HPO₄ 0.3 g/L, MgSO₄.H₂O 0.1 g/L, Ca(NO₃)₂.4H₂O 0.05g/L. Macro-B: KCl 0.5 g/L, KH₂PO₄ 0.2 g/L, MgSO₄.H₂O 0.2 g/L, andCaSO₄.2H₂O 0.2 g/L. Trace Element Stock Solution: H₂BO₃ 1.0 mg/L,ZnSO₄.7H₂O 1.0 mg/L, CuSO₄.5H₂O 0.5 mg/L, MnCl₂.4H₂O 0.5 mg/L,Na₂MoO₄.2H₂O 0.1 mg/L, and Fe-EDTA 1.0 mg/Liter.

Microbial solutions of the present inventions, Sumagro 1-4, were thenprepared using Mineral solution (MM) as a carrier. In some formulations,MM was added at the same volume as HG, described herein for directcomparative experiments. As such, comparative experiments were donewhere duplicate formulations were prepared and used at equal volumeswhere HG was the carrier in one case and MM is the carrier in theparallel treatment. The results obtained with F2 using MM as the carrierwere comparable to those of F2 with HG as the carrier. Therefore, theinventors concluded that these preliminary results showed that HG wasnot an essential ingredient (carrier) and that it can be replaced withsuitable alternative carriers, such as a mineral solution.

b. Microbial Formulations with Nitrogen-Free Mineral Salt Solutions(Sterilized):

CoCL₂.6H₂O 0.004 mg, H₃BO₃ 2.86 mg, MnCL₂.4H₂O 1.81 mg, ZnSO₄.7H₂O 0.22mg, CuSO₄.5H₂O 0.08 mg, H₂MoO₄.H₂O 0.09 mg, MgSO₄.7H₂O 492.96 mg, K₂HPO₄174.18 mg, KH₂PO₄ 136.09 mg, CaCL₂ 110.99 mg, FeC₆H₅O₇.H₂O 5.00 mg, anddistilled water up to 1000.00 ml. pH was adjusted to 6.8 as needed withsterile NaOH and HCl. (Reference: Canadian Food InspectionAgency-Fertilizers-Methods for testing legume inoculants, in Methods forTesting Legume Inoculant and Pre-Inoculated Seed Products FertilizersAct, Section 23, Regulations, PLANT PRODUCTION DIVISION, Canadian FoodInspection Agency, Ottawa, Ontario, K1A 0Y9, Canada, Latest Revision:May, 2005, www.inspection.gc.ca/english/plaveg/fereng/legumee.shtml,herein incorporated by reference).

A broad range of test plants were used in the following greenhousegrowth experiments and field trials. These plants included but were notlimited to a broad spectrum of plants including legumes, cereals,noncereals, vegetables, and forage crops Zea mays (corn), Sorghumbicolor (sorghum), Glycine max (soybean), Phaseolus vulgaris (gardenbean), Pisum sativum (pea), Phaseolus sp. (wonder bush bean), Arachishypogea (peanut), Oryza sativa (rice), Lycopersicon esculentum (tomato),Solanum melongena (eggplant), Hibiscus esculentus (okra), and Cucurbitamaxima (squash), cow pea (Vigna sinensis), green gram (Vigna radiata),black gram (Vigna mungo) zucchini (Cucurbita pepo) and a variety ofgrasses as shown herein.

EXAMPLE II Greenhouse Experiments Using Sumagro 1 and 2

The relative efficacy of Sumagro 1 and Sumagro 2 were tested in labscale experiments done in a greenhouse with standard controlledtemperature and humidity. NutraGro, a commercially available microbialproduct with claimed plant growth enhancing properties was used inparallel on duplicate pots of plants for comparison. No exogenousnitrogen fertilizer or pesticides were added in these experiments.

Procedures:

Sumagro 1 (F1) and Sumagro 2 (F2) described in the preceding sectionswere used. F1 and F2 contained 1014 microbes per ml. For Sumagro 1, onepart of F1 was added to 4 parts of Humate carrier (12% humic acid inwater as mentioned above). For Sumagro 2, one part of F1 was added to 4parts of Humate carrier (12% humic acid in water as mentioned above).Pots (size 4″×5″ and 500 g soil volume) were used for the initialGreenhouse experiments. If the experiment is continued beyond twomonths, then the plants were transferred to pots with 9″ diameter and12″ depth. Enough water was added to just keep the soil moist(approximately 150 ml for the small pots).

Pots prepared as above were used to plant the seeds [or seedlings]depending on the crop. Tomato, eggplant, and okra seeds were germinatedin pot mix soil using 4″×5″ inch pots and 15-day-old seedlings fromthese pots were used for transplantation. For each pot, 4seeds/seedlings were planted and for each treatment two randomizedreplications were made.

Initial Greenhouse treatments included the following: 1. F1 plusHumagro; 2. F2 plus Humagro; 3. Nutragro plus Humagro; and 4. Humagro.

Seeds of all legumes and cereals were imbibed in double distilled waterfor 12 hr before soaking them in various formulations for 30 min. justbefore sowing.

Concentration of Formulations to add per pot was calculated for 500 gramof soil in each pot. Each pot received 4 ml of a formulation and thisgave approximately 10¹⁴ organisms per each treatment. [Note: Theinventors diluted and tested this formulation at a 10¹⁰ organism pertreatment that demonstrated similar growth enhancement results ontreated plants]. One week after planting, all purpose fertilizer with anN:P:K of 15:30:15 (300 mg/pot) was added to each pot as calculated basedon blanket recommendation for fertilizers. Pots were watered to maintainadequate moisture levels.

Two applications of the treatments were given: one at the time ofplanting and the second one 30 days after sowing/planting. A pipette wasused to introduce the formulations at the base of the stems. Theduration of the experiment extended up to three months in some cases.Water was added as needed to maintain 80% field moisture capacity.Parameters used for evaluating the efficacy of the formulations includedany changes in: seed germination, seed emergence, height of the plant,leaf area, shoot length, root length, nodulation (legumes only), shootweight, root weight, time for flowering, and fruiting and diseaseincidence. Parameters were measured at approximately four-weekintervals.

Plants used in Experiment 1 was started in the growth chamber undercontrolled conditions of temperature and humidity. The pots were smalland contained 40 g of soil. These plants were then transferred to largerpots for placement in the greenhouse. At the time of transfer, plantswere transferred to larger pots containing 500 g of soil. Theformulations tested were 1. A=HG+F1; 2. B=HG+F2; 3. C=HG+NG; and 4.D=CONTROL (NO TREATMENT). Abbreviations: HG—Humagro; F1—Sumagro 1:F2—Sumagro 2; NG—Commercial microbial formulation. For each treatmentthere were two replications. The crop plants tested were 1. Corn; 2.Purple hull-pea; Tomato; Brinjal (eggplant); Soybeans; Wonder bushbeans; Garden pea; Zucchini; and Squash plants.

Experiment 1 results demonstrated plant growth enhancer capability ofboth F1 and F2 formulations of the present inventions. In particular,the greatest growth enhancement was seen of purple hull-pea, tomato,brinjal (eggplant), soybean, wonder bush bean, and garden pea plantstreated with F2 (Formulation 2) F1, such that these plants were tallerand/or bushier than similar plants treated with F1 (formulation 1),which showed more plant enhancement than Nutragro, which was in turnslightly better than plants treated with Humagro or control plantstreated with water, see, FIGS. 2-10, especially after 2 months ofgrowth, see FIG. 4. Specifically, plant growth was assessed by plantheight and total leaf area of the plants. FIGS. 2-10 clearly demonstratethe greater performance of Formulation 2 and 1 over Nutragro on similarplants. Early flowering and more number of flowers were also observedwith wonder bush beans.

Plants used in Experiment 2 were seeded and grown under greenhouseconditions from the time of sowing. In Experiment 2, a ‘Humagro only’control was also included in addition to the four treatments shown inExperiment 1. Further, rice, sorghum, okra and peanuts plants wereincluded in addition to the plants tested in Experiment 1.

Experiment 2 results further demonstrated plant growth enhancingcapabilities of F1 and F2, as in Experiment 1, such that growth ofplants treated with either F1 or F2 outgrew, either in height, width orboth, those plants treated the Nutragro and or Humagro (no additives),see, exemplary FIGS. 11-14.

Greenhouse Evaluation Experiments.

Baccto premium potting soil (Michigan peat Company, Houston, Tex.) wasused for growing the selected test plants in the greenhouse experiments.A randomized replicated design was used to set up growth experiments fortesting the efficacy of F1 and F2 formulations. For each 12″×12″×12″pot, two split applications of the liquid formulations (10¹⁰ cfu perpot) were given during the crop period. The first application was givenas soil treatment at the time of sowing and the second application wasgiven at the base of the plant one month after the first application.The experiments were set up in such a way to compare the efficacy of F1and F2 formulations in comparison to a control (HG) containing 12% humicacid alone without any added microbes. Hence, 3 treatments, i.e. F1, F2,and control (HG), each with 4 replications were tested. Exogenousfertilizers or pesticides were not added to any of the three treatmentsduring the crop period. The majority of inoculant standards contain aminimum number of viable microbial cells of at least 10⁹ rhizobia/gramsoil (Brockwell and Bottomley, 1995; Xavier et al. 2004; hereinincorporated by reference). Plant minerals (minus N) were added to eachtreatment 15 days after germination. A broad spectrum of crops whichincludes cereals, vegetable crops, legumes, forage grasses and alsobiofuel grasses were tested. Plants including garden beans, wonder bushbeans, purple hull beans, pea, cowpea, green gram, black gram, soybean,tomato, eggplant, okra, squash, zucchini (Cucurbita pepo), corn,sorghum, rice, and peanut were tested to compare the efficacy of F1 andF2 in enhancing productivity. Observations were made at monthlyintervals during the entire crop period. In a separate experiment, theefficacy of F1 and F2 on germination and growth of commerciallyavailable forage legumes seed mixture (Tecomate Monster Seed Mix, ToddValley Farms, Nebraska) was tested. Plant height, total number ofleaves, leaf area, leaf color, flowering time, fruiting time, shoot androot biomass, and the incidence of pests and diseases were monitored.

The results (Table 6, FIG. 21 to 24) showed a significant increase inplant height with F2 treatment followed by F1 and control. For example,when compared to controls, corn height increased by 65%; egg plant 41%;wonder bush beans 40%; tomato 91%, soybeans 96%, pea purple hull 50%,and okra by 16%. Yield also significantly increased in F2 treatment. Forexample, mean yield of tomato increased by 88% as compared to thecontrol. Okra yield increased 50% and rice increased by 40%. With rice,both F1 and F2 showed an increase in seedling vigor, plant height,number of tillers and their carry over effect on grain yield. Legumestested showed early flowering and fruiting, good root nodulation, and nodisease was observed in both the experimental and control plants duringthe crop period.

There is a significant commercial interest in products thatsubstantially increase productivity of forage crops. The present resultsfurther confirm that F2 formulation enhances the growth of a commercialseed mixture of forage crops, i.e. Tecomate Monster Mix, clover andswitch grass, as compared to humate alone as control (FIGS. 25-27).

TABLE 6 Greenhouse evaluation of polymicrobial formulations F1, F2, andcontrol (C). Plant Height [cm] Yield [g] Crop F2 F1 C F2 F1 C Corn 142125 101.2 — — — Sorghum 74 68.5 49 — — — Rice 65 60 55 20.85  15.76  5.2Tomato 77 72 66 1900*    755*    380 Soybeans 167.7 160.5 98 11.58* 7.9 5.1 Pea 45 38 33 13.99* 10.48* 7.52 Okra 130 93.7 98 138.7*  100*   38.7 Peanut 42 42 35 21.62* 14.67* 6.48 Pea 60.96 46.48 40.64 14.75*12.23* 10.75 purple hull Garden 135 128 102 48.6*  42.6*  23.5 beansWonder 88.9 76.2 63.5 72.9*  63.6  35.6 bush beans Squash 57 41 36650*    230*    0 Significant, P = 0.022

EXAMPLE III Field Trials Demonstrating Plant Growth Enhancement ofSumagro 1 and Sumagro 2

The primary objective was to compare the efficacy of the microbialgrowth enhancement formulations of the present inventions with one ofthe microbial products, claimed to be a plant growth enhancer, such as aproduct claimed to be a plant growth enhancer that is already incommercial use. The commercial product selected for comparison wasNutragro, see above.

Field Trial Applications (Procedure):

Step 1. Concentrated Sumagro 1 (F1) and Sumagro 2 (F2) as described inthe preceding sections were prepared and used for these trials, suchthat F1 and F2, respectively, contained 10¹⁷ and 10¹⁵ microbes per ml.

Step 2. One liter of concentrated F1 or F2 was added to 4 liters ofHumagro (a commercial humic acid carrier) and this 5 liter F1 Mix or F2Mix was further mixed with irrigation water and applied to one acre ofland.

At least 2 applications were applied by irrigation water for one cropseason.

Step 3. A first application was applied to soil at the time of sowingthe seeds. Second application was given before flowering (approximately30 days after sowing, depending on the crop). The inventors recommendedapplication of the formulation at the base of the plants so that theformulation can infiltrate into the soil more effectively in proximityto the plant's root system. A third application (when given) was appliedas a foliar spray approximately 30 days after flowering.

Standard agronomical practices such as appropriate soil tilth, pH,irrigation, and a low level of fertilizer (such as N:P:K of 10:10:10),etc. were adhered to during the field experiments.

Step 4. The field experiments were designed according to a standardrandomized block design with buffer zones of 2 feet on either side ofeach block, in part to prevent edge effects of spray drift. Eachtreatment block of a 10 feet square block was duplicated, such that 2replicates per treatment were provided. The treatments included thefollowing four: 1. F1; 2. F2; 3. Nutragro; and 4. Humagro.

Test crops treated with formulations of the present inventions includedvegetable plants, such as tomato, brinjal, okra, squash, and zucchini;legume plants, such as beans, pea; cereal plants, such as rice, corn,and sorghum; fodder crop plants, such as alfalfa, Bermuda grass, andclover; fiber crop plants, such as cotton, and oil seed plants, such aspeanut plants.

Step 5. Main test parameters were evaluated to compare the efficacy ofF1, F2 and controls, including seed germination (percent), height of theplant, equivalent leaves for leaf area measurements, shoot length, rootlength, nodulation (legumes only), shoot weight, root weight, time forflowering, and fruiting and disease incidence (if any). Parameters asdescribed herein, were evaluated at day 30 and at day 60 after sowing.

Field Evaluation Experiments.

Field trials were conducted with the cooperation of BioSoil Enhancers(Hattiesburg, Miss.) to test the efficacy of the polymicrobialformulations on soybean, corn, cotton, yellow squash, tomato, greenbeans, bell pepper (Capsicum annuum) and banana pepper (Capsicum spp.).The yield data obtained in field trials were consistent with results ofgreenhouse experiments in showing a distinct increase in yield of allthe crops tested. For example, crops treated with polymicrobialformulation F2 showed 75% increase in yield for tomatoes; 27% for bellpeppers; 40% for banana peppers; and 61% for yellow squash (Table 7).Increase in corn yield was 30.0% and cotton plants treated with thepolymicrobial formulation also showed increased plant height, goodbranching, and large sized healthy bolls when compared to control(results not shown). Both greenhouse and field trials indicate thatappropriately formulated polymicrobial formulations have excellentpotential to enhance productivity of a broad spectrum of crops.Moreover, the need for nitrogen fertilizers and pesticides greatlydecreased, which substantially contribute to the conservation of soilhealth, and conservation of fossil fuel energy sources. Further researchprogress in this area would be a substantial contribution to boostingcrop production compatible with sustainable agriculture practices.

TABLE 7 Field Evaluation of Polymicrobial Formulations. F1 F2 - %increase F2 formulation formulation Control in yield Crops (oz peracrea) (oz) (oz) over control Squash 1559 1414 963 61 Tomato 836 514 47775 Banana Pepper 35 15 25 40 Bell Pepper 102 87 80 27

The estimated cost per acre for use of these products is less than $1.00per acre. No exogenous nitrogen fertilizer or pesticides were added inthese experiments.

EXAMPLE IV

This example demonstrates the effects of F2, F3 and F4 on root noduleformation. Unless specified, the soil was not sterilized. However forcertain experiments the inventors grew plants in sterilized soil inorder to demonstrate the endogenous characteristics of the microbes ofthe formulations of the present inventions, such as nodule formationcapabilities of the microbial formulation. Soil was sterilized byautoclave until test samples showed that no endogenous growth wasobserved after watering and observation.

The inventors provided solutions of F2, F3 (a Rhizobial bacterialMixture) and F4 (consisting of Trichoderma strains) nodulationexperiment. The results demonstrated numerous root nodules in bean plantroots during F2 treatment, FIG. 15D, as compared to the few nodulesobserved on untreated bean plant roots, FIG. 15E. These nodules wereshown to be the direct result of microbes in the F2 formulation whencompared to the greatly reduced number and variety of nodules in beanplants grown in sterilized soil (FIG. 15C).

The inventors provided solutions of F2, F3 (a Rhizobial bacterialMixture) and F4 (consisting of Trichoderma strains) nodulationexperiment. The results demonstrated numerous root nodules in bean plantroots during F2 treatment, FIG. 15D, as compared to the few nodulesobserved on untreated bean plant roots, FIG. 15E. These nodules wereshown to be the direct result of microbes in the F2 formulation whencompared to the greatly reduced number and variety of nodules in beanplants grown in sterilized soil (FIG. 15C).

Because the inventors observed increased root nodule formation inleguminous plants treated with an F2 formulation of the presentinvention, see, above, the inventors further treated legume pea plantswith formulations where the microbes were either bacterial or fungalmicrobes in order to separate the bacterial and fungal contributions.

Therefore, the inventors provided a formulation consisting of theSymbiotic Diazotroph bacteria described herein, and a formulationconsisting of five Trichoderma species of the present inventions (F4)Ensifer meliloti FD, Rhizobium trifolii FD, Azorhizobium caulinodans KN,Rhizobium phaseoli CA, and Bradyrhizobium japonicum.

The results further demonstrated Trichoderma specific induction ofnodulation on the roots of garden beans (see, F3 induced nodulation inFIG. 15A compared to F4 induced nodulation in 15B) and pea plants F3induced nodulation as demonstrated in FIGS. 28A and F4 in FIG. 28B. See,Table 8 below.

TABLE 8 Results of root nodulation in garden beans treated with eitherbacterial microbes or fungal microbes. Treatment Nodulation SymbioticDiazotrophs** + Yes* HG Trichoderma + HG Yes* HG only None *Nodulationobserved with diazotrophs (nitrogen-fixating) are larger and morenumerous, and different from those seen with Trichoderma treatment.**Formulated as described herein.

Sumagro 3 and Sumagro 4 were prepared in a sterilized nitrogen-freemineral salts solution (N-free Mineral solution) as described herein.Plants were seeded and treated in pots containing 500 g of sterilevermiculite plus potting soil, in a ratio of 75:25. There were 4 plantsper each pot. Treatments included 1. control (water); 2. N-free Mineralsolution (120 ml of N free solution), 3. F3 plus N-free Mineral solution(4 ml F3 plus 120 ml of N free solution), 3. F4 plus N-free Mineralsolution (4 ml F4 plus 120 ml N-free Mineral solution), and 4. HG(Humagro).

Seeds were soaked in respective treatments for 1 hour prior to sowing.The duration of the experiment was 4 weeks. Nodules on the roots ofplants in a given treatment were recorded. The Figures show roots ofgarden beans treated for 30 days, both, 2×, soaking in 4 ml 30 minutesfollowed by planting, remaining formulation was added to soil at site ofseeding.

Nodulation seen with F3 (Rhizobial Mixture) and F4 (Trichoderma mixture)formulations. Note the clear nodulation seen with F4 (Trichodermamixture) alone. This Trichoderma-dependent nodulation was determined inthe absence of nodulation without Trichoderma under otherwise identicalconditions in similar plants.

Enhanced roof nodulation was observed in all the legumes treated withthe formulations as compared to the controls. The inventors concludedthat Trichoderma-induced root nodule formation by native soil bacteria.

EXAMPLE V

This example demonstrated the plant growth enhancing effects offormulations of the present inventions, specifically two types ofSumagro 2 (F2) formulation each prepared with a different type ofcarrier solution, on a mixture of grass plants grown under greenhouseconditions. As shown, the inventors further demonstrated enhanced growthof plants with Sumagro 2 comprising a mineral solution as a carrier inplace of the Humagro carrier solution used in prior Examples.

Specifically, grass plant seed mixtures were soaked for 30 minutes intheir treatment solution, seeded in greenhouse pots and further treatedwith F2 formulated in Humagro (HG), F2 formulated in the mineralsolution (NF2), as compared to similar grass plants treated with HG andgrass plants treated with mineral solution (MM), as described herein.FIGS. 24A and B shows an exemplary effect of HG vs. F2 (in HG) vs. MMvs. NF2 (F2 in Mineral solution and no HG). Note the increase in growthof grass plants in NF2 that is identical to F2.

These results show that F2 and NF2 stimulate an increase in the growthand thus productivity of a mixture of grasses. Further, these resultsdemonstrate that a mineral solution is an effective carrier solution forformulations of the present inventions.

EXAMPLE VI

This example demonstrated the plant growth enhancing effects offormulations of the present inventions on a variety of switchgrassplants in Greenhouse evaluations.

The inventors planted grass seeds of the following varieties for testingwith the F2 formulation as described in Example 1. Control pots weremerely treated with water. While the varieties showed some enhancedgrowth, 27A) Carthage and 27C Dacotah (Dakota), while 27B) Cave-in-Rockand more significantly 27D) Forestburg showed significantly enhancedgrowth when fed F2 formulation of the present inventions.

Further contemplated are additional varieties for use, singly or asmixtures, with biomass increasing formulations and/or biocontrolformulations of the present inventions including but not limited to“Trailblazer, Sunburst, Summer, Shelter=NY4006, REAP 921, Pathfinder,Pangburn, Nebraska 28, Kanlow, Forestburg, Carthage=NJ-50, Caddo,Blackwell, Alamo, et cetera.

EXAMPLE VII

This example demonstrated the biological control effects of isolates ofthe present inventions for inhibiting the growth of phytopathogenicmicrobes.

The inventors discovered that F2, which contains fungal isolates of thepresent inventions shown to be active against pathogenic fungalisolates, provided protection against powdery mildew. Further, F4 (afungal strain mixture) provided plant protection to a variety ofpathogens, including but no limited to Curvularia lunata (leaf spot oftomato; Fusarium solani (tomato wilt); Bipolaris oryzae, (brown leafspot of rice); Magnoporthe grisea, (blast disease of rice); Alternariaalternata (early blight of tomato and potato), and Rhizoctonia solani(Sheath blight of rice) as well as the plant-pathogenic bacterium,Xanthomonas oryzae (bacterial blight of rice). In particular, all theTrichoderma species isolated herein were screened for their biocontrolpotential against known plant pathogenic fungi using the dual platetechnique.

Isolates of the present inventions were tested for biocontrol effects,including but not limited to inhibiting the growth of phytopathogenicfungal species. In particular, isolates of Trichoderma harzianum, T.viridi, T. longibrachiatum, T. virens, and the bacteria Pseudomonasfluorescens, were tested as possible biocontrol agents of Alternariaalternata and Curvularia Sp. causing leaf spot of tomato under in vitroconditions. T. harzianum showed dominance and hyper parasitism oncontact over A. alternata and Curvularia sp. T. virens, T.longibrachiatum and T. viridi also inhibited and hyper parasitized A.alternata. Biocontrol was governed by different mechanisms such ascompetition for space and nutrients, mycoparasitism, and possibleantibiosis.

Inhibition of plant pathogens—Dual culture plate method. In thisExample, four different species of Trichoderma that are present in. F2were tested against Alternaria alternata, an important pathogen oftomato plants. As shown in exemplary FIG. 29A, where Alternariaalternata would typically grow over an entire plate of agar (plate inlower left of A), each of the three fungal isolates tested causedinhibition of growth of the pathogenic fungus. Such that TH Trichodermaharzianum; TV T. virile; and TL T. longibrachiatum were designated bythe inventors as Bio-control fungus. T. virens, was also tested againstAlternaria alternata and found to have anti-fungal properties. Further,a B5 bacteria strain, Pseudomonas fluorescens of the present inventionswas shown to have anti-growth effects on several types of pathogens,including A Alternaria alternata (Tomato leaf spot pathogen); CCurvularia sp. (Tomato leaf spot pathogen); F Fusarium solani (Tomatopathogen) as shown in FIG. 29B.

Greenhouse observations. During the course of formulation testing underGreenhouse conditions, the inventors observed plants infected withpowdery mildew caused by Microsphaera diffusa. Surprisingly, infectedplants in adjacent pots showed significant resistance to infection whengrown in the presence of F2. Specifically, exemplary comparisons aredemonstrated in FIG. 30A where soybean plants 1-3 were treated withconventional fertilizer while plant 4 was undergoing F2 treatment.Plants 1-3 show large white spots indicative of infection while plant 4is essentially free of fungal spots. Even more striking were squashplants with numerous blooms undergoing F2 treatment showing signs offungal infection on larger (older) green leaves while a control squashplant is wilted and almost dead following symptoms of Powdery mildewinfections.

Thus F2-treated plants are highly resistant to Powdery mildewinfections.

EXAMPLE VIII

This example provides an exemplary method for growing mixtures ofmicrobes for long-term storage. Modifications of this method arecontemplated for providing deposits under the Budapest Treaty.

The inventors grew individual bacteria isolates listed for Sumagro 5,Table 2C (also the bacterial portion of the Sumagro-2 mixture) accordingto methods provided herein. These isolates were mixed together to form abacterial mixture (labeled F2A) in Sumagro-2 which is combined with themixture of fungal isolates (labeled F2B) described below. Further, a F2Amixture is contemplated for shipment to the NRRL for deposit under theBudapest Treaty as NRRL accession B-50215.

Similarly, the fungal isolates listed for Sumagro 4, Table 4, (also thefungal portion of the Sumagro-2 mixture) were grown in the inventor'slaboratory, described supra. These isolates were mixed together to forma fungal isolate mixture (labeled F2B) for use in combination with F2A.Further, a F2B fungal isolate mixture is contemplated for shipment tothe NRRL for deposit under the Budapest Treaty as NRRL accession 50216.

An exemplary reference for culture preservation and re-growth of thebacterial and fungal isolates is provided, Gherna, R. L. and C. A.Reddy. 2007. Culture Preservation, p 019-1033. In C. A. Reddy, T. J.Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R.Snyder, eds. American Society for Microbiology, Washington, D.C., 1033pages; herein incorporated by reference.

We claim:
 1. A microbial formulation, wherein said formulation comprisesa microbial mixture of a bacterial mixture having N.R.R.L accessionnumber B-50215 and a fungal mixture having N.R.R.L accession number50216.
 2. The microbial formulation of claim 1, further comprising, aliquid carrier.
 3. The microbial formulation of claim 2, wherein saidliquid carrier comprises water and humic acid.
 4. The microbialformulation of claim 3, wherein said humic acid is at a concentration of12% volume of humic acid (ml)/volume of solution (ml).
 5. The microbialformulation of claim 2, wherein said liquid carrier has a pH of 7.0. 6.The microbial formulation of claim 2, wherein said microbial mixture hasa microbe concentration in the liquid carrier that ranges from 10¹⁰-10¹⁷microbes per milliliter of liquid.
 7. The microbial formulation of claim1, wherein the formulation is selected from the group consisting of aliquid, a dried formulation, and a wettable powder.
 8. The microbialformulation of claim 7, wherein the dried formulation is a freeze driedliquid formulation.
 9. The microbial formulation of claim 1, whereinsaid formulation is a biocontrol formulation for a pathogen selectedfrom the group consisting of leaf spot of tomato and powdery mildew. 10.The microbial formulation of claim 9, wherein said pathogen comprises aCurvularia species and an Alternaria species.
 11. The microbialformulation of any of claim 10 and 2-7, further comprising an adjuvantselected from the group consisting of a wetting agent, spreading agent,dispersing agent, sticking agent, or adhesive.